title: Investigating the role of free fatty acid receptor 4 in 3T3-L1 Adipocytes
Creators: name: Alshammari, Woroud Sayel
Publisher: name: University of Glasgow
Year: 2023

description: FFA4 is a G protein coupled receptor activated by long chain fatty acids that has been found to inhibition of inflammation, improve insulin sensitivity and adipogenesis, and regulate hormone secretion from the gastro-intestinal system and pancreatic islets. All these reports indicate the strong relationship between maintaining metabolic homeostasis and FFA4, which puts this receptor as a great potential target to treat obesity and type 2 diabetes. In adipocytes, FFA4 expression increasing during adipocyte differentiation and there is some evidence that inhibiting FFA4 reduces adipogenesis. However, there are still gaps in understanding FFA4 and its function in adipocytes. In particular, most studies have relied on either natural fatty acid to activate the receptor, or genetic approaches to reduce receptor expression. Currently there is a lack of research using pharmacological ligands to understand FFA4 function in adipocytes. The current study is aimed to address this by assessing the effects of FFA4 agonism and antagonism on a 3T3-L1 in vitro model of adipocytes. Cells were treated with either an FFA4 agonist, TUG-891, or an FFA4 antagonist, AH7614.

Initially, pharmacological assays were performed in cell lines that heterologously express mouse ortholog of FFA4 to confirm the pharmacological properties of TUG-891 and AH7614. TUG-891 was found to induce phosphorylation of extracellular signal regulated kinase, elevate Ca+2 mobilization, increase levels of β-arrestin recruitment and induce receptor internalisation. On the other hand, AH7614, effectively inhibited both TUG-891 and fatty acid agonists of FFA4 in calcium mobilisation, β-arrestin2 recruitment, and internalisation assays. This inhibition appeared to be non-competitive antagonism.

3T3-L1 cells were used as platform for functional experiments to uncover the effects of FFA4 on glucose uptake and lipolysis in mature adipocytes. While acute TUG-891 treatment of 3T3-L1 adipocytes had no significant effect on glucose uptake, AH7614 treatment significantly decreased glucose uptake. In lipolysis assays, a range of concentration of TUG-891 significantly inhibited βadrenoceptor stimulated lipolysis, while different concentrations of AH7614 increased this lipolysis.

Observation of the acute effects of FFA4 agonism and antagonism on 3T3-L1 adipocytes inspired me to next explore the effects of TUG-891 and AH-7614 treatment during the adipogenesis process in 3T3-L1 cells. 3T3-L1 adipocytes that were differentiated in the present of TUG-891 or AH7614 went through assays to estimate the effect of agonist and antagonist treatment in adipogeneses. Oil Red O staining revealed that AH7614 significantly reduced lipid content, while TUG-891 had limited effect. This was supported by RT-qPCR showing that AH7614 treatment significantly decreased expression of various adipogenic genes including: FFA4 itself, PPARγ, adiponectin, leptin and GLUT4. In contrasts, while TUG-891 did significantly increase FFA4 expression, it had no effect on the other adipogenic genes. 3T3-L1 adipocyte function in cells differentiated in the presence of TUG-891 and AH7614 was assessed in two ways: by glucose uptake upon insulin stimulation and by measurement of isoprenaline stimulated lipolysis. Consistent with the observed effects on lipid uptake and gene expression, AH7614 treatment during differentiation reduced both insulins stimulated glucose uptake and isoprenaline stimulated lipolysis in the mature adipocytes, while TUG-891 had no significant impact on either readout.

These finding highlighted the role of FFA4 in adipocyte since its activation is curial for adipogenesis. Moreover, the ability of AH7614 to reduce in 3T3-L1 adipocyte raises the possibility of using FFA4 antagonists in the fight against obesity. Yet this suggestion could be complicated, since this FFA4 antagonists also inhibited glucose transport. Together, this thesis uses pharmacological tools to provide new insight into the function of FFA4 in adipocytes and the potential for targeting this receptor in the treatment of metabolic disorders.