The unique role of Ces3 in PPAR-mediated thermogenesis

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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.