The unique role of Ces3 in PPAR-mediated thermogenesis

脂滴是一种胞质细胞器，在大多数细胞类型中充当主要的能量储存库。 脂肪组织低密度脂蛋白中中性脂的过度积累与肥胖及肥胖相关代谢紊乱有关。LDs是动态形成、放大和收缩的，这种动态受其表面结合的多个蛋白质的调节。最近，我们发现羧酸酯酶3(Ces3)能响应β-肾上腺素能信号刺激而移位到LDs表面。我们发现，阻断LDs上的CES3不仅降低了脂解作用，而且还削弱了β-肾上腺素能信号刺激的产热作用。这项研究的目标是确定整个过程的管理机制。根据我们的初步观察，我们假设LD靶向的CES3产生脂肪酸(FFA)作为特异性PPARγ配体(S)来触发脂肪组织的产热。为了检验这一假设，我们提出了三个具体目标。在目标1中，我们将研究Ce3在LDS上的移位机制(S)。我们将首先确定cAMP-PKA信号如何调节Ces3在LDS上的转位。然后，我们将研究Perilipin-1在Ces3转位到LDS中的作用。在目标2中，我们将研究Ces3产生的游离脂肪酸在生热中的作用。为此，我们的初步结果显示 循环中的二十二碳六烯酸(DHA，C22：6n=3)，一个已建立的PPARγ配体，是戏剧性的 在脂肪组织特异性Ces3基因敲除小鼠中降低。因此，我们的研究将集中在产生DHA的信号机制上，DHA是CES3内源性PPARγ的关键配体。我们将首先确认CES3在体外产生DHA，并测试β-肾上腺素能信号刺激是否增加DHA的水平。然后我们将检测与PPARγ结合的DHA是否也增加。最后，我们将对CES3生产的DHA的功能进行表征。在目标3中，我们将评估缺乏Ces3的脂肪细胞的脂代谢失调。我们将首先检测Ces3功能丧失时包括DHA在内的脂质产物的变化。我们将通过药物和遗传工具来抑制Ces3的活性，并通过定量脂组学来评估游离脂肪酸(FFA)的变化。同时，我们将测试对PPARγ转录活性和UCP-1介导的生热程序的影响。然后，我们将研究脂肪细胞中缺乏Ces3时LDS的失调。特别是，我们将检查胞浆LDS在CES3消融后的形态变化。我们将进一步测试经典脂肪酶轴ATGL-HSL-MGL是如何受到影响的。这项拟议的研究旨在从LDS的动态调节角度阐明一种将脂肪代谢与能量消耗联系起来的新途径。我们的研究将解决CES3及其产物DHA在调节代谢稳态中的关键作用。调查结果将突出这些新的参与者在治疗肥胖症和肥胖相关疾病方面的巨大潜力。

Lipid droplets (LDs) are cytosolic organelles that serve as the major energy reservoir in most cell types. Excessive accumulation of neutral lipids in LDs of adipose tissue has been linked to obesity and obesity-associated metabolic disorders. LDs are dynamically forming, enlarging as well as shrinking and the dynamics is regulated by multiple proteins bound to their surfaces. Recently, we identified Carboxylesterase 3 (Ces3) that translocates onto the surface of LDs in response to β-adrenergic signaling-stimulation. We showed that blockage of Ces3 on LDs led to not only decreased lipolysis, but also impaired β-adrenergic signaling-stimulated thermogenesis. The goal of this study is to define the mechanisms governing the whole process. Based on our preliminary observations, we hypothesize that the LD-targeting Ces3 generates fatty acids (FFA) “third messenger” that serve as specific PPARγ ligand(s) to trigger thermogenesis in adipose tissue. To test this hypothesis, we propose three specific Aims. In Aim 1, we will study the mechanism(s) by which Ces3 translocates onto LDs. We will first determine how cAMP-PKA signaling regulates the translocalization of Ces3 onto LDs. Then, we will investigate the role of Perilipin-1 in the translocalization of Ces3 onto LDs. In Aim 2, we will study the function of FFAs generated by Ces3 on thermogenesis. For this Aim, our preliminary results reveal that circulating Docosahexaenoic acid (DHA, C22:6 n=3), an established PPARγ ligand, is dramatically decreased in adipose tissue-specific Ces3 knockout mice. Our study will thus focus on a signaling mechanism that underlie the production of DHA - a key endogenous PPARγ ligand by Ces3. We will first confirm production of DHA by Ces3 in vitro and test whether DHA level is increased by β-adrenergic signaling-stimulation. Then we will detect whether DHA bound to PPARγ is increased as well. Finally, we will characterize the functions of DHA produced by Ces3. In Aim 3, we will assess the dysregulation of lipid metabolism in Ces3-deficient adipocytes. We will first exam the changes of lipid productions including DHA upon loss-of-function of Ces3. We will inhibit Ces3 activity by both pharmacological and genetic tools and assess the changes of free fatty acids (FFA) by quantitative lipidomics. Meanwhile, we will test the effects on the PPARγ transcriptional activity and UCP-1 mediated thermogenic program. Then, we will study the dysregulation of LDs at the absence of Ces3 in adipocytes. Particularly, we will exam the morphological changes of the cytosolic LDs upon Ces3 ablation. We will further test how the classical lipase axis ATGL-HSL-MGL is affected. The proposed study aims to elucidate a novel pathway that links lipid metabolism to energy expenditure from the viewpoint of dynamic regulation of LDs. Our study will address the critical roles of Ces3 and its product DHA in regulation of metabolic homeostasis. Results from the investigation will highlight the great potential of these novel players for therapeutic application in obesity and obesity-associated diseases.