Oscillations in Oxygen Consumption by Permeabilized Clonal Pancreatic β-Cells (HIT) Incubated in an Oscillatory Glycolyzing Muscle Extract: Roles of Free Ca²⁺, Substrates, and the ATP/ADP Ratio

To determine whether oscillations in glycolysis could underlie the oscillations in O₂ consumption observed in intact islets, we evaluated the capacity of an islet extract to exhibit spontaneous oscillations in glycolysis. When a cell-free extract obtained from ∼1,000 islets was supplied with glucose and glycolytic cofactors, oscillations in NADH fluorescence were obtained. After this demonstration of spontaneous oscillations in islet extracts, we bathed permeabilized clonal β-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of α-glycerophosphate and pyruvate and induces oscillations in free Ca²⁺ and the ATP/ADP ratio. This preparation was used to investigate whether changes in Ca²⁺ and possibly α-glycerophosphate or pyruvate supply could underlie observed oscillations in O₂ consumption and explain coordination between cytosolic and mitochondrial metabolism. We found that oscillations of O₂ consumption and Ca²⁺ of a similar period were induced. Removal of medium Ca²⁺ with EGTA did not prevent the oscillations in O₂ consumption nor were they greatly affected by the substantial rise in medium Ca²⁺ on treatment with thapsigargin to inhibit sequestration into the endoplasmic reticulum. The O₂ oscillations were also not eliminated by the addition of relatively high concentrations of pyruvate or α-glycerophosphate. However, they were lost on addition of fructose-2,6-P₂ at concentrations that prevent oscillations of glycolysis and the ATP/ADP ratio. Addition of a high concentration of ADP increased O₂ consumption and also prevented O₂ oscillations. These results suggest that the changes in respiration reflected in the O₂ oscillations occur in response to the oscillations in the ATP/ADP ratio or ADP concentration and that this parameter is a primary regulator of O₂ consumption in the pancreatic β-cell.