Investigating methods of assessing the biological function of a novel sucralose receptor, GPR52

Abstract

The effects of non-nutritive sweeteners are increasingly controversial, with research suggesting harmful metabolic and physiological effects. Previous research found sucralose activates an orphan GPCR, GPR52. Orphan GPCRs lack known functions and endogenous ligands and are thus lacking research tools to establish their biological function. The aim of this study was to develop tools to study the biological function of GPR52. GPR52 expression in metabolically relevant organs in rats was first confirmed by PCR. Three research tools were then explored: lentiviral gene knockdown, qPCR, and pharmacological tools. Gene knockdown can be achieved via RNAi delivered through a lentiviral expression system. Lentiviral plasmid DNA was transfected into cells with shRNA targeting GPR52. After optimizing plasmids and cell health, the cells produced lentivirus expressing the shRNA, confirmed by the expression of GFP. Functional lentivirus was transduced onto cells to knock down GPR52 expression, confirmed by GFP expression. qPCR primers were designed and validated by determining primer efficiency. The expression of GPR52 was measured in biologically relevant cell lines, as well as a control of HEK293T cells transfected with GPR52 DNA. The qPCR showed off-target amplification and the formation of primer dimers when visualized via agarose gel electrophoresis. Pharmacological tools like agonists can be used to modulate ligand binding and receptor activation. A novel GPR52 agonist, FTBMT, was used to activate GPR52, and was measured via the PRESTO-Tango assay. FTBMT treated cells showed morphological changes consistent with cell death, and the receptor was not activated. Cell viability when exposed to FTBMT was measured via MTT assay, which showed no relationship between cell viability and FTBMT treatment. The development of these tools is crucial to the study of the biological functions of GPR52 and could provide insight on the effects of sucralose on metabolism mediated through the receptor.