Background and aims: Increasing the efficacy of GLP-1 receptor (GLP-1R) agonists is currently the main challenge in type 2 diabetes research. GLP-1R is a G protein-coupled receptor (GPCR) known to be positively coupled to cAMP production and PKA/EPAC2 activations and to recruit the scaffold protein beta-arrestin2 (ARRB2), which may activate new signaling pathways such as the kinases ERK1/2. Most of the studies have investigated GLP-1R signaling in recombinant clonal non-beta cell lines. Consequently, a better understanding of the mechanisms involved in GLP-1R internalization/desensitization and signaling in primary pancreatic beta cells is necessary. In addition, special attention should be paid to the effect of therapeutic pharmacological concentrations found in type 2 diabetes patients treated with GLP-1R agonists (nM) versus circulating physiological concentrations (pM) of GLP-1. Materials and methods: Experiments were performed in beta cells from 4-month-old Arrb2-/- and Arrb2+/+ male mice. PKA (AKAR3) and ERK1/2 (EKAR) activities, and EPAC2 recruitment underneath the plasma membrane (EPAC2-GFP), were assessed by live-cell microscopy in mouse pancreatic beta cells after genetic expression of the sensors of interest by adenoviral infection. ERK1/2 (P-ERK1/2) and CREB (P-CREB) activation were assessed by immunofluorescence. GLP-1R internalization was determined by immunofluorescence from 4% formaldehyde fixed and non-permeabilised beta cells. Results: In beta cells, GLP-1 (10pM to 10nM) caused a rapid activation of PKA, which remained sustained during stimulation (>40min). This activation persisted (~25 min) after cessation of GLP-1 stimulation at pharmacological concentrations (slope -0.4649±0.04) but not at physiological concentrations (slope -0.8826±0.11, p=0.001). Interestingly, the recovery was faster with a direct activator of the adenylate cyclase (1µM forskolin; slope: -2.673±0.10; p<0.001) that trigger larger activation of PKA. In parallel, EPAC2 is activated at pharmacological (10nM) and not physiological (10pM) concentrations of GLP-1. This was associated with massive GLP-1R internalization (~60%, p<0.001), which remained 25 min after cessation of stimulation (~40% p<0.001) at pharmacological concentrations. In contrast, a lack of GLP-1R internalization was observed at physiological GLP-1 concentrations (10-100pM). ARRB2, known to uncouple GPCRs from G protein and induce their internalization, is not involved in this process. Finally, pharmacological concentrations led to sustained activation of ERK1/2 kinases and nuclear activation of CREB only in the presence of ARRB2 (p<0.001). In contrast, physiological concentrations of GLP-1 caused transient ARRB2-independent activation of ERK1/2 and do not activate CREB. Conclusion: This study reports for the first time that physiological and pharmacological concentrations of GLP-1 resulted in distinct cellular spatiotemporal responses in primary beta cells. Special attention should be paid to cell signaling when generating new GLP-1R agonists to treat type2 diabetes.