The unique role of Ces3 in PPAR-mediated thermogenesis

脂滴（LDs）是胞质细胞器，在大多数细胞类型中充当主要的能量储存器。 中性脂质在脂肪组织LD中的过度积累与肥胖和肥胖相关的代谢紊乱有关。LD动态地形成、扩大和缩小，其动态受到与其表面结合的多种蛋白质的调节。最近，我们鉴定了羧酸酯酶3（Ces 3），其响应于β-肾上腺素能信号刺激而易位到LD表面。我们发现，阻断LD上的Ces 3不仅导致脂解减少，而且还损害了β-肾上腺素能信号刺激的产热。本研究的目的是确定整个过程的机制。基于我们的初步观察，我们假设LD靶向Ces 3产生脂肪酸（FFA）“第三信使”，其作为特异性PPARγ配体触发脂肪组织中的产热。为了验证这一假设，我们提出了三个具体目标。在目标1中，我们将研究Ces 3易位到LD上的机制。我们将首先确定cAMP-PKA信号如何调节Ces 3在LD上的转位。然后，我们将研究Perilipin-1在Ces 3转定位到LD中的作用。目的2：研究Ces 3产生的游离脂肪酸在产热过程中的作用。对于这个目标，我们的初步结果显示， 循环中的二十二碳六烯酸（DHA，C22：6 n=3），一种公认的PPARγ配体， 在脂肪组织特异性Ces 3敲除小鼠中降低。因此，我们的研究将集中在一个信号传导机制，基础上的生产DHA -一个关键的内源性PPARγ配体的Ces 3。我们将首先在体外证实Ces 3产生DHA，并测试DHA水平是否因β-肾上腺素能信号刺激而增加。然后我们将检测DHA与PPARγ结合是否也增加。最后，我们将描述由Ces 3产生的DHA的功能。在目标3中，我们将评估Ces 3缺陷脂肪细胞中脂质代谢的失调。我们将首先检查Ces 3功能丧失后脂质产生的变化，包括DHA。我们将通过药理学和遗传学工具抑制Ces 3活性，并通过定量脂质组学评估游离脂肪酸（FFA）的变化。同时，我们还将检测其对PPARγ转录活性和UCP-1介导的产热程序的影响。然后，我们将研究脂肪细胞中Ces 3缺失时LD的失调。特别地，我们将检查Ces 3消融后胞质LD的形态学变化。我们将进一步测试经典脂肪酶轴ATGL-HSL-MGL如何受到影响。 该研究旨在从LD动态调节的角度阐明一种将脂质代谢与能量消耗联系起来的新途径。我们的研究将解决Ces 3及其产物DHA在调节代谢稳态中的关键作用。调查结果将突出显示这些新型参与者在肥胖和肥胖相关疾病治疗应用中的巨大潜力。

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.