title: Investigating the physiological functions of free fatty acid receptor 4 in the lung and colon following synthetic agonist activation
Creators: name: Euston, Eloise
Publisher: name: University of Glasgow
Year: 2023

description: Free fatty acids not only function as dietary nutrients but act as signalling molecules by activating the free fatty acid family of G protein-coupled receptors. Following deorphanisation in 2005, FFA4 was found to be abundantly expressed in the colon where it was suggested to play a role in GLP-1 incretin secretion. This has been the basis for the development of synthetic FFA4 agonists as treatment for type 2 diabetes mellitus. Although more recently, the role of FFA4 in the lung, a tissue which also show high levels of FFA4 expression, has been investigated. Within the lung, FFA4 has been shown to exert anti-inflammatory effects in lung resident macrophages, as well as mediating airway smooth muscle relaxation. Therefore, FFA4 represents an attractive drug target for both metabolic diseases and inflammatory lung diseases. However, there is a substantial lack of FFA4 agonists reaching clinical trials due to a limited number of available agonists, issues with selectivity of drugs and poor pharmacokinetic and pharmacodynamic properties, which highlights the need for greater interest in the development and scientific research of these receptor agonists. This thesis aimed to extend on previous work performed in our lab, exploring the roles of FFA4 receptor agonists in airway smooth muscle relaxation and the previously undefined role that phosphorylation may play in airway smooth muscle relaxation. Additionally, the involvement of FFA4 in GLP-1 incretin secretion is a somewhat controversial topic, with reports suggesting that FFA4 is a primary mechanism in GLP-1 secretion and opposing reports suggesting that FFA4 has no involvement in the role of GLP-1 secretion from the colon. This thesis also aimed to answer the question of whether GLP-1 release is mediated by FFA4 activity. To characterise signalling mechanisms for the mouse FFA4 receptor, functional assays were performed on cell lines which stably express the mouse ortholog of FFA4. Here it was confirmed that FFA4 primarily couples to Gαq/11 G proteins, with no evidence of Gαs or Gαi coupling in cell lines. Additionally, bias factor calculations were performed which indicated that carboxylic agonist, Agonist 2, displayed bias towards IP1 signalling pathways. Similarly, signalling mechanisms of phosphorylation deficient (PD) mouse FFA4 were also characterised using the same functional assays. Here, agonist activation of the PD-mFFA4 receptor induced enhanced IP1 and pERK1/2 signalling but resulted in a loss β-arrestin2 coupling and receptor internalisation. Functional bias calculations revealed that several agonists acting on the PD-mFFA4 receptor display bias towards IP1 signalling, revealing a complex nature of signalling downstream of Gq:receptor coupling. Functional experiments to assess the physiological roles of FFA4 in the lung and colon revealed some interesting results. Quantification of mouse lung airway diameter following FFA4 agonist addition to pre-contracted airways indicated a robust relaxation of airway smooth muscle. Furthermore, in a novel finding, there was an indication that airway relaxation was mediated in part by receptor phosphorylation. Additionally, to understand the contribution of FFA4 in GLP-1 release from the colon, experiments assessing GLP-1 release from primary colonic crypts following FFA4 agonist treatment were performed. Following 50 μM agonist treatment, GLP-1 secretion was significantly increased, exposing another physiological role of the FFA4 receptor. These findings validate FFA4 as a novel drug target in the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease and also in the treatment of metabolic diseases such as diabetes. Current drug therapies for these diseases can result in harmful side effects and may be ineffective on certain populations of patients, highlighting a clinical need for novel and safer therapeutics. Thus, research on novel FFA4 drugs is crucial to bring new drugs to clinic.