Potential health promoting properties of the triterpenoid ursolic acid as a putative mimetic of caloric reduction - studies in cultured cells and in mice

熊果酸（UA）是一种植物性五环三萜。我们自己之前的工作表明，UA改善了果蝇的健康和寿命，并改善了与年龄相关的运动活动下降。这一发现与Spargel （srl）表达增加有关。Spargel是哺乳动物过氧化物酶体增殖物激活受体γ共激活物（PGC1）的同源物。然而，尚不清楚我们在果蝇中的数据是否可以转化为哺乳动物物种。因此，我们将解决UA的潜在延长寿命特性是否也存在于哺乳动物中以及它们是否通过PGC1活性的改变而介导的问题。为此，我们将采用三种（I-III）实验方法。由于Pgc1被认为是UA介导作用的潜在关键因素，我们将通过siRNA和CRISPR-Cas9沉默Pgc1α的表达，检测UA在小鼠肌肉和肝细胞中孵育后Pgc1α靶基因的表达情况。因此，我们将在体外证明Pgc1α可能参与UA介导的生物活性(I)。其次，我们将在Pgc1α敲除小鼠模型中研究Pgc1α靶基因在选定组织（肌肉、肝脏）、原代肌细胞和肝细胞中的表达对膳食UA的响应。UA将与γ-环糊精（γCD）络合以提高其生物利用度。因此，我们将在体内验证Pgc1α可能参与UA介导的效应（II）。CR诱导Pgc1α表达，随年龄增长而下降。因此，我们将再次研究UA对老年小鼠Pgc1α表达的作用，以及在自由或限制补充UA-γ cd的饮食中对健康和寿命的影响。我们将反对ua衍生的结果与CR，并说明任何潜在的协同或添加性生物活性。因此，我们打算澄清UA是否可以被视为潜在的CR模拟物（III）。广泛地说，在所有三种实验方法中，我们将确定Pgc1α靶基因的活性，编码线粒体功能（通过oroboros呼吸测量法）和代谢中重要蛋白质的基因的活性，关键因素和选定的效应分子以及miRNA的活性。我们将通过ChIP分析、硅对接分析和报告基因分析更详细地验证和表征UA介导的结果。这些参数将通过小鼠的综合表型来补充，包括身体组成、间接量热、口服葡萄糖耐量、肌肉力量和运动活动。

Ursolic acid (UA) is a phytogenic pentacyclic triterpenoid. Our own previous work shows that UA improves health and life span in the fruit fly Drosophila melanogaster and ameliorates the age-related decline in locomotor activity. This finding is referred to an increase in Spargel (srl) expression. Spargel represents the orthologue of mammalian peroxisome proliferator-activated receptor gamma coactivator (PGC1). However, it is unclear whether our data in flies can be translated into mammalian species. Thus, we will address the question whether the potential life-prolonging properties of UA also exists in mammals and whether they are mediated through a change in PGC1 activity. To this end, we will follow three (I-III) experimental approaches. As Pgc1 is considered as the potential key factor for UA-mediated action, we will examine the expression of Pgc1α target genes after UA incubation in murine muscle and liver cells with siRNA and CRISPR-Cas9 silenced expression of Pgc1α. Hence, we will demonstrate a potential involvement of Pgc1α in UA mediated bioactivity in vitro (I). Secondly, we will investigate the expression of Pgc1α target genes in selected tissues (muscle, liver) and primary myocytes and hepatocytes in a Pgc1α-knock-out mouse model in response to dietary UA. UA will be complexed with γ-cyclodextrin (γCD) to increase its bioavailability. Consequently, we will verify a potential involvement of Pgc1α in UA mediated effects in vivo (II). Pgc1α expression is induced under CR and declines with age. Therefore, we will thirdly study the role of UA on Pgc1α expression and on health and life span in old mice providing a UA-γCD-supplemented diet ad libitum or restricted. We will oppose the UA-derived results to CR and illustrate any potential synergistic or additive bioactivity. Thus, we intent to clarify whether UA could be considered as a potential CR mimetic (III). Extensively, in all three experimental approaches, we will determine the activities of Pgc1α target genes, of genes encoding proteins important in mitochondrial function (via Oroboros-respirometry) and metabolism, of key factors and of selected effector molecules as well as miRNA. We will validate and characterize the UA mediated outcome in more detail via ChIP assay, in silico docking analysis and reporter gene assays. These parameters will be complemented by a comprehensive phenotyping of mice including body composition, indirect calorimetry, oral glucose tolerance, muscle strength and locomotor activity.