Figure 3. Analysis of the effect of a novel compound in HEK293T cells overexpressing GPR21.

The direct effect of GRA2 on IP₁ production was assessed using a functional FRET-based IP-one assay with HEK293T cells transiently transfected with (a) pCMV6-Entry or GPR21 and (b) pCMV6-Entry or GPR21 coupled with Gα_15/16. To analyse the downstream effects of GRA2 on regulating the impact of GPR21 on the insulin signalling pathway, HEK293T cells overexpressing (c) pCMV6-Entry or GPR21 and (d) pCMV6-Entry or GPR21 coupled with Gα_15/16 were incubated with 10 μM GRA2 or an equal volume of the vehicle control in serum free medium for 24 h followed by a 1 h stimulation with 100 nM insulin. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with antibodies against phospho-Insulin Receptor β Tyr^1150/1151, Insulin Receptor β, phospho-IRS1 Tyr⁶¹², IRS1, phospho-Akt Ser⁴⁷³, Akt, phospho-AS160 Thr⁶⁴², phospho-AS160 Ser⁵⁸⁸, AS160, myc and β-actin. Western blots are representative of two separate experiments. To establish the direct impact on glucose uptake, incorporation of [³H]-2-deoxyglucose was evaluated via scintillation counting of solubilised cells overexpressing (e) pCMV6-Entry or GPR21 and (f) pCMV6-Entry or GPR21 coupled with Gα_15/16. Data presented as mean ± SEM are representative of two independent experiments performed in triplicate. A significant increase in glucose uptake was observed at p < 0.05*, p < 0.01** and p < 0.001***.