Pharmacology of novel free fatty acid receptor 4 ligands and their potential use in metabolic studies

Abstract

Free fatty acids serve as both dietary nutrients and signalling molecules by activating the G protein-coupled receptors of the free fatty acid family. After being deorphanized in 2005, free fatty acid receptor 4 (FFAR4) was shown to be highly expressed in the pancreas, where it was proposed to have a function in the production of insulin. The development of synthetic FFAR4 agonists as a potential therapy for type-2 diabetes mellitus has been founded on this. Recently, there has been research on the function of FFAR4 in the pancreas, specifically in δ-cells, which have high levels of FFAR4 expression. FFAR4 has been shown to be expressed in various types of islet cells, such as α, β, δ, and γ cells, inside the pancreas. FFAR4 plays a crucial role in regulating insulin and glucagon synthesis, as well as suppressing the release of somatostatin in the islets of Langerhans. Thus, FFAR4 serves as a compelling pharmaceutical target for metabolic disorders. Nevertheless, the lack of FFAR4 agonists in clinical trials is primarily attributed to a smaller number of available agonists, challenges with drug selectivity, and suboptimal pharmacokinetic and pharmacodynamic characteristics. This underscores the necessity for increased attention and scientific investigation into the development of these receptor agonists. The objective of this thesis was to conduct a pharmacological characterization of new FFAR4 ligands and evaluate their potency and efficacy in comparison to the reference agonist, TUG-891. The research aimed to determine the specific location of FFAR4 in pancreatic islets and examine the effects of FFAR4 expression on the function of these cells. Moreover, the objective of the thesis was also to assess the capacity of FFAR4 ligands to induce insulin secretion from pancreatic islets. This research aims to enhance the understanding of FFAR4's involvement in glucose regulation and its potential as a target for treating metabolic diseases. Moreover, this study aims to enhance the knowledge of FFAR4 pharmacology and its physiological roles in the pancreas. By doing so, it will provide vital insights for the creation of new treatment approaches that specifically target this receptor. In order to analyse the signalling processes of the mouse FFAR4 receptor, functional tests were conducted on cell lines that constitutively express the mouse ortholog of FFAR4. It has been verified that FFAR4 mostly associates with Gαq/11 G proteins, and there is little or no indication of coupling with Gαs or Gαi in cell lines. Out of the ligands tested, FFAR4 Agonist II showed greater efficacy, whereas Merck cpd A and GSK137647A revealed similar or lower efficacy compared to TUG-891, which was used as the standard ligand. Based on the analysis, it was shown that the FFAR4 Agonist II is a superior ligand compared to TUG-891. FFAR4 Agonist II has the potential to be a useful tool to conduct experiments both ex vivo and in vivo to validate FFAR4 as a viable target for treating metabolic diseases. Functional tests have shown that FFAR4 plays a pivotal function in the regulation of hormone production in the pancreas. Although the FFAR4 ligand TUG-891 has a minor impact on the release of insulin from β-cells, FFAR4 is crucial for enhancing insulin secretion caused by the M3 agonist oxotremorine. This effect was not observed in FFAR4- KO islets. Interestingly, the combination of TUG-891 with oxotremorine and FFAR4 antagonist AH7614 resulted in a 2.5-fold reduction in the impact of oxotremorine. In addition, the phosphorylation of the FFAR4 receptor seems to have a role in insulin release. This was shown by comparing the response of islets from a mutant mouse line expressing an FFAR4 variation that is defective in phosphorylation (PD mouse) to wild-type islets when exposed to oxotremorine. In addition, somatostatin release was 2-3 times higher in FFAR4- KO islets than in wild-type islets, demonstrating that FFAR4 regulates somatostatin secretion independently of ligand activation. The results highlight the potential of FFAR4 as a target for treating metabolic diseases. These findings confirm that FFAR4 is a new and promising target for therapeutic development in the treatment of metabolic diseases, namely T2DM. Existing treatments for T2DM, such as metformin, may lead to adverse effects and may not be successful for specific patient groups. This emphasises the need for new and safer medications in clinical practice. The capacity of FFAR4 to regulate the production of insulin and somatostatin in the pancreas, together with its ability to control glucose homeostasis, emphasises its therapeutic promise. Additional investigation into the precise mechanisms that control FFAR4 activation and signalling pathways has the potential to result in the creation of targeted medications that successfully regulate glucose metabolism and enhance patient outcomes while reducing the adverse effects associated with current medicines.