The cells were plated in 35-mm dishes and stimulated with 100 nM exendin-4 for 3 h. The supernatant was collected, and the cells were lysed in RIPA buffer. Insulin content in the supernatant and lysate was http://www.selleckchem.com/products/Dasatinib.html calculated according to the manufacturer’s protocol. Ubc-9 knock down was accomplished using a validated short-hairpin RNA (shRNA) vector (Origene). Retroviral particles were generated in HEK-292 cells according to the manufacturer’s instructions (Clontech, Mountain View, CA). Four constructs were tested for efficiency in MIN6 cells, and one showing ~50% knock-down efficiency was selected. Dispersed mouse islets were infected for 16 h and cultured in 16 or 5 mM glucose for 24 h. The islet cells were stimulated with 16 mM glucose and 50 nM exendin-4 for 2 h, and secreted and cytosolic insulin was quantified by ELISA (ALPCO).

Data Analysis The statistical analysis was carried out by Graphpad Prism software. RESULTS Mouse Islets Exposed to High Glucose Show Increased Expression of SUMO and Ubc-9 Transcripts Expression of the components of the SUMO pathway is upregulated by various environmental stress conditions such as osmotic, hypoxic, heat, oxidative, and genotoxic stresses (36). Here, we investigated whether chronic exposure of islets to high glucose elicits a similar response. Mouse islets incubated in 16 mM glucose for 48 h showed increased expression of SUMO-1 (6.4-fold), SUMO-2 (4.3-fold), SUMO-3 (1.7-fold), and Ubc-9 (3.8-fold) compared with islets in 5 mM glucose (Fig. 1). This result shows that exposure of pancreatic islets to high glucose concentrations results in an increase in expression of SUMO and Ubc-9 transcripts.

Fig. 1. Expression of small ubiquitin-related modifier protein (SUMO) transcripts in mouse islets. Quantitative real-time RT-PCR shows increased expression of SUMO-1 transcripts in mouse islets exposed to high glucose. The fold change in expression of SUMO transcripts … Enhanced Expression of SUMO-1 Downregulates GLP-1R Activation of the GLP-1R signaling pathway stimulates adenylate cyclase, resulting in an increase in cytosolic cAMP. Therefore, the amount of free cAMP in the cell following agonist treatment should be a function of receptor activity assuming no change in PDE activity (10). The dynamic changes in cAMP concentration due to sumoylation were studied using a FRET-based biosensor.

FRET between CFP and YFP fused to the cAMP-binding domain of the exchange Batimastat protein Epac-2 (Epac-camps) was used to measure spatial and temporal changes in cAMP signaling (17). The Epac biosensor undergoes conformational changes upon cAMP binding that result in loss of FRET, measured as an increase in FRET ratio. MIN6 cells expressing endogenous GLP-1R were transiently cotransfected with Epac-camps and mCherry-tagged SUMO-1 or mCherry vector.