Endocrine Abstracts

Background: G-Protein Coupled Receptors-120 (GPR120) and GPR40 are of interest as targets for treating diabetes and obesity due to their involvement in incretin secretion. This study investigated the mechanistic function of dual agonism on regulating the regeneration of beta cell mass, proliferation, and the insulin secretory response, using CRISPR/Cas9 gene knockout cells and intracellular-signalling analysis.

Methods: Clonal pancreatic BRIN-BD11 cells and GPR120 K/O cells were treated with GPR120/GPR40 agonists to determine GPR120 and GPR40 expression by qPCR. Protein expression was assessed by immunocytochemistry; insulin secretion by radioimmunoassay at normoglycaemic and hyperglycaemic conditions; and cell toxicity by MTT analysis. Phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT1/2/3) were investigated in BRIN-BD11/GPR120 K/O cells by western blotting.

Results: qPCR demonstrated that the stimulatory effect of GPR120/GPR40 agonist Alpha-Linolenic Acid (A-LA) was completely abolished in GPR120 K/O cells, no GPR120 gene expression was observed at 5.6mM (P < 0.001) and 16.7mM (P < 0.001) glucose. Addition of A-LA resulted in upregulation of GPR40 gene expression in GPR120 K/O cells (P < 0.05) at 16.7mM glucose. Immunocytochemistry confirmed co-localisation of insulin/GPR120 in BRIN-BD11 cells, and lack of GPR120 expression in K/O cells. Agonists A-LA, Compound A and GSK137647 increased insulin secretion in a dose-dependent manner in BRIN-BD11 cells (P < 0.05- P < 0.001), with no cytotoxicity. Western blot showed GSK137647 increased AKT1/2/3 expression at 16.7mM glucose (P < 0.05). Dual agonism by A-LA resulted in upregulation of phosphorylated ERK1/2 in BRIN-BD11 cells (by 31%, P < 0.05) at 5.6mM glucose and a 54% reduction in expression (P < 0.01) in GPR120 K/O cells. A-LA had no effect on GLP-1R gene expression in K/O cells confirming the importance of GPR120/GPR40 agonism.

Conclusion: Dual activation of GPR120/GPR40 has an important role in regulating pancreatic islet growth and proliferation, mediating anti-apoptotic effects, and has significant potential for new drug development for diabetes treatment.