No-flow ischemia inhibits insulin signaling in heart by decreasing intracellular pH

Glucose-insulin-potassium solutions exert beneficial effects on the ischemi c heart by reducing infarct size and mortality and improving postischemic l eft ventricular function. Insulin could be the critical protective componen t of this mixture, although the insulin response of the ischemic and postis chemic myocardium has not been systematically investigated. The aim of this work was to study the insulin response during ischemia by analyzing insuli n signaling. This was evaluated by measuring changes in activity and/or pho sphorylation state of insulin signaling elements in isolated perfused rat h earts submitted to no-flow ischemia. Intracellular pH (pH(i)) was measured by NMR. No-flow ischemia antagonized insulin signaling including insulin re ceptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, protei n kinase B, p70 ribosomal S6 kinase, and glycogen synthase kinase-3. These changes were concomitant with intracellular acidosis. Perfusing hearts with ouabain and amiloride in normoxic conditions decreased pH(i) and insulin s ignaling, whereas perfusing at pH 8.2 counteracted the drop in pH(i) and th e inhibition of insulin signaling by ischemia. Incubation of cardiomyocytes in normoxic conditions, but at pH values below 6.75, mimicked the effect o f ischemia and also inhibited insulin-stimulated glucose uptake. Finally, t he in vitro insulin receptor tyrosine kinase activity was progressively inh ibited at pH values below physiological pH(i), being abolished at pH 6.0. T herefore, ischemic acidosis decreases kinase activity and tyrosine phosphor ylation of the insulin receptor thereby preventing activation of the downst ream components of the signaling pathway. We conclude that severe ischemia inhibits insulin signaling by decreasing pH(i).