Background and aims: Increasing the efficacy of GLP-1 receptor (GLP-1R) agonists by using dual GLP-1/GIP receptor agonist is a new strategy in the treatment of type 2 diabetes. GLP-1R and GIPR are G protein coupled receptors (GPCRs) known to be positively coupled to cAMP production and PKA/EPAC2 activations. Both GPCRs are also able to recruit the scaffold protein beta-arrestin2 (ARRB2), which may activate new signaling pathways such as the kinases ERK1/2 or Focal Adhesion Kinase (FAK). Our aim is to determine whether Tirzepatide, a dual GLP-1/GIP receptor agonist, has a beneficial effect superior to GLP-1 on primary pancreatic beta cells. Materials and methods: Experiments were performed in beta cells from 4-month-old Arrb2-/- and Arrb2+/+ male mice. Endogenous PKA (AKAR3) and ERK1/2 (EKAR) activations were assessed by live cell imaging in mouse pancreatic beta cells after adenoviral infection with FRET-based sensors of interest. Insulin secretion was measured on isolated islets by homogenous time-resolved fluorescence. F-actin depolymerization was evaluated by phalloidin staining (Alexa Fluor 488-conjugated phalloidin) and the phosphorylation of FAK by immunofluorescence. GLP-1R endocytosis was assessed by immunofluorescence from 4% formaldehyde fixed and non-permeabilised beta cells. Results: PKA activation and insulin secretion were significantly increased in response to 10pM-100pM GLP-1 (p<0.01) and maximally activated by 100nM-1nM. In contrast, GIP and Tirzepatide were only effective from 1nM (p<0.01), indicating, that Tirzepatide is less potent than GLP-1 on GLP-1R, as already reported in non-beta cells. In addition, GLP-1 induced PKA long lasting activation (>25 min) that was associated with a prolonged internalisation of GLP-1R, whereas the reversal of PKA activation upon GIP stimulation was rapid (<5min), suggesting slow versus fast recycling receptors, respectively. Surprisingly, Tirzepatide induced PKA long lasting activation similar to that of GLP-1, but without any GLP-1R internalisation. In Arrb2-/- beta cells, PKA and EPAC2 activations by GLP-1 or GIP were not affected despite a strong reduction of insulin secretion in response to GIP (~50%, p<0.01) that was caused by a reduction of F-actin depolymerisation (~50%, p<0.01) and FAK activation (~50%, p<0.01). Tirzepatide, like GLP-1, induced a similar insulin secretion, F-actin depolymerisation and FAK activation in Arrb2+/+ and Arrb2-/- beta cells. By contrast, ERK1/2 activation in response to GLP-1 was strongly reduced by ~50% (p<0.05) in Arrb2-/- beta cells while GIP and Tirzepatide recruited ERK1/2 independently of ARRB2. Conclusion: In beta cells, Tirzepatide combines the beneficial effects of GLP-1 and GIP. Our study reports that this dual GLP-1/GIP agonist may overcome the functional consequences of the decrease in ARRB2 expression that can be observed in diabetogenic conditions.