Wortmannin

Roscovitine (Calbiochem), wortmannin (LC Laboratories), and BX795 (Enzo Life Sciences) were dissolved in DMSO at concentrations of 50, 20, and 6 mM, respectively, as stock solutions and used at final concentrations of 45, 40, and 6 µM, respectively.

All animal procedures were performed according to the guidelines provided by the Committee of Animal Ethics at Korea University. Fifteen healthy male 4-week-old Sprague Dawley rats (Orient, Technical Corporation of Charles River Technology, Gapyoung, Korea) weighing 150±10 g were randomly divided into 3 groups: the control, diabetic and wortmannin-treated diabetic groups (n=5 for each group). Before the experiments, the rats were allowed 1 week to adapt to the laboratory conditions. The rats were maintained under standard conditions with a 12 h/12 h light/dark cycle at 23±1 °C and 50±10% humidity. All animals had free access to water. All animals in the diabetic and wortmannin-treated diabetic groups were intraperitoneally injected with 65 mg kg⁻¹ body weight streptozotocin (STZ; Sigma-Aldrich, St Louis, MO, USA) dissolved in pH 4.5 sodium citrate buffer. Control rats were injected with an equivalent volume of sodium citrate buffer. At 3 days after STZ injection, the animals were considered to be diabetic if the plasma glucose levels were ⩾300 mg dl⁻¹.^{23 (link)} wortmannin (1 mg kg⁻¹, LC Laboratories) was dissolved in dimethyl sulfoxide and intraperitoneally injected every day for 8 weeks. The control and diabetic group rats were injected with an equivalent volume of dimethyl sulfoxide. All rats were killed after 8 weeks.^{17 (link)}

The TRPM3 agonist PregS and TRPM8 agonist menthol were purchased from Sigma-Aldrich. ATP was purchased from Roche. The nonhydrolyzable ATP analogue adenosine-5-[(β,γ)-methyleno]triphosphate (AMPPCP) was obtained from Jena Bioscience and adenosine-5-[(β,γ)-imido]triphosphate (AMPPNP) was purchased from Sigma-Aldrich. Water-soluble diC8 PIPs and diC16 PI(4,5)P₂ were purchased from Echelon, IP₃ was obtained from Sigma-Aldrich, and brain-derived natural PI(4,5)P₂ was obtained from Avanti. Stock solutions of PIPs were reconstituted in H₂O and stored at −80°C, and they were intensively sonicated before use. Estimated mole fractions in the inner leaflet of the membrane for diC8- PI(4,5)P₂ and diC8- PI(4)P were calculated based on the polynomial functions provided in Collins and Gordon (2013) (link), which provide a relationship between PIP concentrations in solution and their distribution in artificial liposomes. To scavenge PIPs, poly-L-lysine (PLL) and neomycin were used (both from Sigma-Aldrich). The phosphatidylinositol-3 kinase (PI-3K) inhibitors wortmannin and LY294,002, as well as rapamycin were from LC Laboratories. The mAChR receptors were activated by oxotremorine-M (N,N,N-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide; Oxo-M) from BioTrend Chemicals.