Tissue-Specific Metabolic Reprogramming in Diabetic Complications

描述（由申请人提供）：1型糖尿病（T1DM）改变碳水化合物、氨基酸和脂肪酸代谢，导致肾病、视网膜病变和周围神经病变，这是糖尿病最令人衰弱的三种并发症。普遍的观点认为，全身性高血糖在所有组织中通过相似的生化机制驱动并发症。然而，这还没有经过专门的测试，特别是在体内。在这些研究中，我们提出并发症易发组织细胞的正常细胞营养利用是不同的，因此对胰岛素缺乏、高血糖等T1DM变化的反应也将是不同的。事实上，我们的初步数据表明，细胞代谢物水平的组织特异性变化不是由单独的质量作用驱动的，而是由于目标组织的选择性代谢重编程。与肾脏中糖酵解和三羧酸循环（TCA）中间产物水平升高相反，尽管环境高血糖，糖尿病小鼠神经和视网膜中的糖酵解和三羧酸循环中间产物水平降低。重要的是，小鼠肾脏和人类尿液中TCA代谢物水平的增加预测了糖尿病肾病的进展，这表明代谢重编程可能在并发症的进展中起致病作用。这些发现支持了我们的假设，即糖尿病并发症是由组织特异性代谢重编程引起的，这种重编程导致燃料利用的改变，从而导致组织功能障碍。为了验证这一假设，我们将在糖尿病模型中使用基于质谱的敏感和特异性代谢组学分析来确定三种并发症易发组织（视网膜、肾脏和周围神经）中代谢物水平和通量的变化。我们将把这些研究扩展到T1DM患者，以了解与非糖尿病患者在肾脏代谢物水平和通量方面的内在差异。我们将定义mRNA和蛋白表达的变化以及翻译后蛋白修饰，以确定代谢水平改变的基础。最后，我们将利用适当改造的小鼠动物来直接测试这些研究中产生的精细假设。我们的具体目标是：目标1：使用最先进的代谢组学方法确定糖尿病并发症最佳小鼠模型中肾脏、神经和视网膜中间代谢稳态异常的变化；目标2：确定小鼠模型中所有3种组织中的代谢物通量，并确定导致代谢物异常的关键调节反应。目的3：评估伴有或不伴有微血管并发症的1型糖尿病患者的稳态和动态代谢物变化。目的4：明确易发生并发症组织中细胞代谢改变的调节机制，并在小鼠模型中测试其作用。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1DM) alters carbohydrate, amino acid, and fatty acid metabolism contributing to nephropathy, retinopathy and peripheral neuropathy, three of the most debilitating complications of diabetes. The prevailing view suggests that systemic hyperglycemia drives complications by similar biochemical mechanisms in all tissues. However, this has not been specifically tested, especially in vivo. In these studies, we propose that the normal cellular nutrient utilization in cells of complication-prone tissues is distinct and therefore reaction to insulin deficiency, hyperglycemia and other changes of T1DM will also be distinct. Indeed, our preliminary data demonstrate tissue-specific changes in cellular metabolite levels that are not driven by mass-action alone but appear to be due to selective metabolic reprogramming of the target tissues. In contrast to increased levels in kidney, glycolysis and tricarboxylic acid cycle (TCA) cycle intermediates are decreased in diabetic mouse nerve and retina despite ambient hyperglycemia. Importantly, the increased mouse kidney and human urinary levels of TCA metabolites predict progression of diabetic nephropathy, suggesting that metabolic reprogramming may play a pathogenic role in the progression of complications. These findings lead to our hypothesis that diabetic complications arise from tissue-specific metabolic reprogramming resulting in alterations in fuel utilization which lead to dysfunction of the tissue. To test this hypothesis, we will use sensitive and specific mass spectrometer based metabolomic analysis in models of diabetes to define changes in metabolite levels and flux in three complications-prone tissues, retina, kidney and peripheral nerves. We will extend these studies to humans with T1DM to understand intrinsic differences from non-diabetics in metabolite levels and flux in the kidney. We will define the changes in mRNA and protein expression and post-translational protein modification to determine the basis for altered metabolite levels. Finally, we will utilize appropriately engineered mouse animals to directly test the refined hypotheses arising from these studies. Our specific aims are to: Aim 1: Identify the alterations in steady state abnormalities of intermediary metabolism in kidney, nerve and retina in the best murine models of diabetic complications using state-of-the-art metabolomic approaches Aim 2: Determine metabolite flux in all 3 tissues from the murine models and identify the key regulatory reactions that contribute to the metabolite abnormalities. Aim 3: Assess steady state and dynamic metabolite changes in humans with type 1 diabetes with and without microvascular complications. Aim 4: Define regulatory mechanisms of altered cellular metabolism in complication-prone tissues and test their effect in murine models. PUBLIC HEALTH RELEVANCE: This proposal will test the hypothesis that diabetic complications arise from specific changes in cellular substrate metabolism which can be defined using modern molecular phenotyping techniques in both animal models and in humans and that interventions to modulate specific metabolic pathways may mitigate or prevent development and progression of these complications. Our study is designed to gain a better understanding of the changes in metabolite levels and flux in complications-prone tissues in animal models and patients with type 1 diabetes mellitus and to determine how the metabolite changes reflect altered levels or activities of specific proteins and lipids which contribute to 'microvascular' complications. We will utilize in vivo and in vitro analysis of mouse models and human patients to achieve these goals.