Fig. 2.

Overexpression of SUMO-1 decreases cAMP response following agonist stimulation of glucagon-like peptide-1 receptor (GLPP-1R). MIN6 cells and mouse primary islet cells were transfected with Epac-camps and mCherry-SUMO or mCherry vector. Dynamic changes in cAMP binding were measured as the change in the fluorescent resonance energy transfer (FRET) ratio. A: change in FRET ratio following exposure to 100 nM exendin-4 in MIN6 cells transfected with Epac-camps and mCherry (black) and Epac-camps and mCherry-SUMO (gray). B: change in FRET ratio in mouse primary β-cells transfected with Epac-camps and mCherry (black) or mCherry-SUMO (gray) when treated with 100 nM exendin-4. C: graph showing mean change in FRET ratio in MIN6 cells. Overexpression of SUMO-1 caused 5- and 3.7-fold reduction in FRET ratio compared with the control in MIN6 cells and mouse primary β-cells, respectively. Error bars indicate means ± SD. Student's t-test: ***P < 0.001; n = 6 cells from multiple dishes. D: total cAMP quantified by enzyme-linked immunosorbent assay (ELISA) after exendin-4 treatment of MIN6 cells transfected with green fluorescent protein (GFP) or GFP-SUMO. FAC-sorted GFP-SUMO cells showed only a 1.3-fold increase in total cAMP, whereas control GFP cells showed a 1.9-fold increase compared with untreated cells. A similar response of 1.8-fold increase was also seen in control cells when exendin-4 treatment was done in the presence of 100 μM IBMX, whereas SUMO-1-expressing cells showed only a 1.2-fold increase that is not statistically significant. Error bars indicate means ± SD; n = 6. Student's t-test: P = 0.01–0.05 (*) and <0.0001 (***) for exendin-4-treated cells without IBMX and with IBMX, respectively.