Tissue-Specific Metabolic Reprogramming in Diabetic Complications

描述(申请人提供)：1型糖尿病(T1 DM)改变了碳水化合物、氨基酸和脂肪酸的代谢，导致肾病、视网膜病变和周围神经病变，这是糖尿病最令人虚弱的三种并发症。流行的观点认为，全身性高血糖通过相似的生化机制在所有组织中驱动并发症。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，我们认为易发生并发症的组织细胞对正常细胞营养物质的利用是不同的，因此对胰岛素缺乏、高血糖和其他T1 DM变化的反应也将是不同的。事实上，我们的初步数据表明，细胞代谢物水平在组织中的特定变化不仅仅是由质量作用驱动的，而且似乎是由于目标组织的选择性代谢重新编程所致。与肾脏中糖酵解和三羧酸循环(TCA)循环中间产物的增加相反，糖尿病小鼠的神经和视网膜中的中间产物减少，尽管环境中存在高血糖。重要的是，小鼠肾脏和人类尿中TCA代谢物水平的增加可以预测糖尿病肾病的进展，这表明代谢重编程可能在并发症的进展中发挥致病作用。这些发现导致了我们的假设，糖尿病并发症是由组织特异性代谢重新编程引起的，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敏感和特定的质谱仪进行代谢组学分析，以确定视网膜、肾脏和周围神经这三个易发生并发症的组织中代谢物水平和代谢通量的变化。我们将把这些研究扩展到患有T1 DM的人身上，以了解与非糖尿病患者在肾脏代谢物水平和流量方面的内在差异。我们将定义mRNA和蛋白质表达的变化以及翻译后蛋白质的修饰，以确定代谢物水平改变的基础。最后，我们将利用适当的转基因小鼠动物来直接测试这些研究产生的精炼假说。我们的具体目标是：目标1：利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：从小鼠模型中确定所有三个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：评估伴有和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：明确易发生并发症的组织细胞代谢改变的调节机制，并在小鼠模型中测试其效果。 与公共卫生相关：这项提议将检验这样一种假设，即糖尿病并发症是由于细胞底物代谢的特定变化而产生的，这种变化可以在两种动物模型和人类中使用现代分子表型技术来定义，以及调节特定代谢途径的干预措施可能会减轻或阻止这些并发症的发展和进展。我们的研究旨在更好地了解在动物模型和1型糖尿病患者中易发生并发症的组织中代谢物水平和流量的变化，并确定代谢物变化如何反映特定蛋白质和脂肪的水平或活性的变化，这些变化有助于微血管并发症的发生。我们将利用对小鼠模型和人类患者的体内和体外分析来实现这些目标。

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.