Fatty Acid Signaling via GPCRs in Primary Cilia Controls Adipogenesis and Insulin Secretion, Regulating Obesity and Diabetes

这个项目的重点是了解来自w-3脂肪酸的膳食流量如何激活FFAR4。 受体，在初级纤毛中表达，在前脂肪细胞中促进脂肪生成，在胰腺b细胞中促进胰岛素分泌。这一建议源于杰克逊实验室最近的一项发现，即在新生脂肪形成过程中，初级纤毛和小鼠脂肪块的积累需要戏剧性的要求(细胞2019)。通过敲除TULP3，纤毛GPCR运输的关键调节因子，该小组建立了纤毛GPCR信号的要求，并筛选了36个候选基因，发现w-3脂肪酸(FA)受体FFAR4/GPR120是关键的。使用FFAR4和纤毛标记物，研究小组发现纤毛FFAR4+前脂肪细胞在所有脂肪垫中组成血管周围干细胞群。此外，DHA-FFAR4信号与胰岛素信号协同指导成脂命运的改变，FFAR4激动剂迅速激活纤毛中局部的cAMP。为了更好地确定FFAR4下游的成脂步骤，进行了两个全基因组范围的CRISPR筛查，以寻找在成脂中重要的基因，一个使用传统的成脂配方，另一个由FFAR4激动剂激活。这些屏幕概括了大多数已知要求 并为成脂计划提供了大量重要的新基因。其中，对控制翻译启动和大小写独立翻译的新因素的需求非常强烈。最后，为了研究FFAR4在其他代谢系统中的重要性，研究小组发现，所有类型的胰岛细胞都是纤毛细胞，但纤毛中的b细胞和a细胞都表达FFAR4。详细的药理学和遗传学研究表明，FFAR4激动剂可以在分离的b细胞或胰岛中促进葡萄糖刺激的胰岛素分泌(Gsis)，其水平与已知的促分泌剂如glp-1相当。FFAR4激动剂也能刺激细胞分泌胰高血糖素。这两种效应都需要纤毛交易。第一个目标将集中于验证和解释在筛查中发现的新的脂肪生成因素。这将侧重于识别特定的腺苷酸环化酶、磷酸二酯酶、cAMP效应因子和翻译控制基因。目标2将集中在胰腺b细胞中的FFAR4信号，以了解FFAR4和cAMP如何驱动胰岛素分泌的机制，以及另一种w-3脂肪酸受体FFAR1如何驱动钙内流与FFAR4协同作用。最后，在目标3中，重点是使用系统地敲除FFAR4和FFAR1的小鼠模型来检查这些途径在肥胖、胰岛素分泌和糖尿病中的协同作用。这一目标还将检查人类患者的脂肪组织，以确定在老龄化、糖尿病或肥胖患者中前脂肪细胞中的FFAR4+纤毛是否丢失，并比较性别差异。总体而言，Aim 3将测试FFAR4或纤毛丢失是否是糖尿病疾病进展的重要驱动因素，建立FFAR4和其他GPCR的关键测试，以正常化脂肪生成并改善老年糖尿病患者的胰岛素分泌。

This project focuses on understanding how dietary fluxes from w-3 fatty acids activate the FFAR4 receptor, expressed in primary cilia, in both preadipocytes to trigger adipogenesis and in pancreatic b cells to enhance insulin secretion. The proposal extends from a recent discovery in the Jackson lab of a dramatic requirement for primary cilia in de novo adipogenesis and for the accumulation of fat mass in mice (Cell 2019). Using a knockout of TULP3, a critical regulator of ciliary GPCR trafficking, the group has established a requirement for ciliary GPCR signaling and screened 36 candidates to discover that the w-3 fatty acid (FA) receptor FFAR4/GPR120 is critical. Using FFAR4 and ciliary markers, the group finds that ciliated FFAR4+ preadipocytes comprise a perivascular stem cell population in all fat pads. Further, DHA-FFAR4 signaling synergizes with insulin signaling to direct adipogenic fate change and that FFAR4 agonists rapidly activate localized cAMP in the cilium. To better define the steps in adipogenesis downstream of FFAR4, two genome-wide CRISPR screens for genes important in adipogenesis were conducted, one using a traditional adipogenesis formulation and the other activated by FFAR4 agonists. These screens recapitulated most known requirements for adipogenesis and provided a wealth of new genes important for the adipogenic program. Among these there is a strong requirement for new factors controlling translation initiation and cap-independent translation. Finally, looking for the importance of FFAR4 in other metabolic systems, the group discovered that all pancreatic islet cell types are ciliated, but that both b and a cells express FFAR4 in cilia. Detailed pharmacology and genetics show that FFAR4 agonists can drive glucose stimulated insulin secretion (GSIS) in isolated b cells or islets at levels comparable to known secretagogues like GLP-1. FFAR4 agonists also stimulate glucagon secretion in a cells. Both effects require ciliary trafficking. The first Aim will focus on validating and explaining new adipogenesis factors found in the screen. This will focus on identifying specific adenylate cyclases, phosphodiesterases, cAMP effectors, and translational control genes. Aim 2 will focus on FFAR4 signaling in pancreatic b cells to understand the mechanisms of how FFAR4 and cAMP drive insulin secretion and how another w-3 fatty acid receptor, FFAR1, drives calcium influx to cooperate with FFAR4. Finally, in Aim 3, the focus is to use mouse models systematically knocking out both FFAR4 and FFAR1 to examine the cooperation of these pathways in obesity, insulin secretion and diabetes. This aim will also examine human patient adipose tissue to determine if FFAR4+ ciliary in preadipocytes are lost in aging, diabetic or obese patients, also comparing sex differences. Overall, Aim 3 will test whether FFAR4 or ciliary loss is an important driver of diabetic disease progression, setting up critical tests of FFAR4 and other GPCRs for normalizing adipogenesis and improving insulin secretion in aging, diabetic patients.