Temporal sequence of metabolic and ionic events in glucose-stimulated clonal pancreatic β-cells (HIT)

Stimulation of insulin release by glucose requires increased metabolism of glucose and a rise in cytosolic free Ca²⁺ in the pancreatic β-cell. It is accompanied by increases in respiratory rate, pyridine and flavin nucleotide reduction state, intracellular pH and the ATP/ADP ratio. To test alternative proposals of the regulatory relationships among free Ca²⁺, mitochondrial metabolism and cellular energy state, we determined the temporal sequence of these metabolic and ionic changes following addition of glucose to clonal pancreatic β-cells (HIT). Combined measurements of the native fluorescence of reduced pyridine nucleotides and oxidized flavin, intracellular pH, and free Ca²⁺ were performed together with simultaneous measurement of O₂ tension or removal of samples for assay of the ATP/ADP ratio. The initial changes were detected in three phases. First, decreases occurred in the ATP/ADP ratio (< 3 s) and increases in pyridine (2±1 s) and flavin (2±1 s) nucleotide reduction. Next, increases in the O₂ consumption rate (20±5 s), the ATP/ADP ratio (29±12 s) and internal pH (48±5 s) were observed. Finally, cytosolic free Ca²⁺ rose (114±10 s). Maximal changes in the ATP/ADP ratio, O₂ consumption and pyridine and flavin nucleotide fluorescence preceded the beginning of the Ca²⁺ change. These relationships are consistent with a model in which phosphorylation of glucose is the initial event which generates the signals that lead to an increase in respiration, a rise in the ATP/ADP ratio and finally influx of Ca²⁺. Our results indicate that Ca²⁺ does not function as the initiator of increased mitochondrial respiration.