Figure 3.

M3R-stimulated insulin secretion and second-messenger responses in Gnb5^−/− and Gnb5^+/+ Min6 cells. A) Wild-type Min6 cells were cultured and treated with 20 ng/ml pertussis toxin (PTX), 0.1 μM FR, 10 μM U73122, Ca²⁺-free medium, or 10 μM nifedipine (Nif), and released insulin was measured after stimulation with 100 μM Oxo-M. Data are means ± sd from at least 3 independent experiments. There was no statistically significant effect of PTX. B) MIN6 cells were transduced with the cAMP sensor and imaged in a flow cell on a fluorescence microscope in real time. After 200 s, solutions of 1 μM forskolin, 100 μM Oxo-M, or 100 nM GLP-1 were added for 200 s. Shown is representative results of 3 independent experiments. C) Free Ca²⁺ responses to 100 μM Oxo-M in the knockout (Gβ5 KO) and control MIN6 cells were recorded by using fura-2 with microscopy. Data points are averages from the total of 100–200 cells. Shown is a representative of 3 independent experiments. D) Cells were transduced with the fluorescence sensor for DAG. Data are representative of 2 experiments. E) Effect of ionomycin (Ion) on insulin secretion from the Gnb5^−/− MIN6 cells. Oxo-M (100 μM) and ionomycin (4 μM) were added in the presence of 16.7 mM glucose. Data are means ± sd from at least 3 independent experiments. *P < 0.05; **P < 0.01.