Alkaline pHo activates insulin receptor family members (InsRFs) in the heart.
(A and B) Rat heart or kidney whole extracts (A) or heart fractions from differential centrifugation (B) were immunoblotted for the INSRR α subunit. WH, whole heart. (C and D) HEK293 cells expressing human FLAG-INSRRs were treated (1 min) with insulin (50 mU/ml) or pHo 9.1 buffer with/without the indicated concentrations of linsitinib. Samples were immunoblotted with antibodies to phosphorylated (Phospho-) InsRFs, FLAG or phospho- or total PKB/Akt. Blots are representative of three independent cell preparations. IC50 values for individual experiments were determined from densitometric data (D). Results are means ± SEM (n = 3). *P < 0.05 for IC50 for INSRR activated by alkaline pHo relative to IC50 for the insulin receptor (INSR) activated by insulin (unpaired two-tailed t-test). (E) Male rat hearts were perfused (15 min) with Tris buffer at starting pHo 9.1 or in Krebs Henseleit buffer (KHB) with/without 50 mU/ml insulin. Samples were immunoblotted with antibodies to phospho-InsRFs, or total PKB/Akt. Densitometric analysis is on the right as individual data points with means ± SEM (n = 4 per group). Statistical analysis used one-way ANOVA with Holm–Sidak post-test. Positions of relative molecular mass markers are on the left of the blots. (F) Activation of PKB/Akt by alkaline pHo is mediated via InsRFs. Hearts were perfused as indicated with/without 1 µM linsitinib and samples were immunoblotted for phospho- or total PKB/Akt.