TEMPORAL SEQUENCE OF METABOLIC AND IONIC EVENTS IN GLUCOSE-STIMULATEDCLONAL PANCREATIC BETA-CELLS (HIT)

Stimulation of insulin release by glucose requires increased metabolis m of glucose and a rise in cytosolic free Ca2+ in the pancreatic beta- cell. It is accompanied by increases in respiratory rate, pyridine and flavin nucleotide reduction state, intracellular pH and the ATP/ADP r atio. To test alternative proposals of the regulatory relationships am ong 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 beta-cells (HIT). C ombined measurements of the native fluorescence of reduced pyridine nu cleotides and oxidized flavin, intracellular pH, and free Ca2+ were pe rformed together with simultaneous measurement of O-2 tension or remov al of samples for assay of the ATP/ADP ratio. The initial changes were detected in three phases. First, decreases occurred in the ATP/ADP ra tio (< 3 s) and increases in pyridine (2 +/- 1 s) and flavin (2 +/- 1 s) nucleotide reduction. Next, increases in the O-2 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). M aximal changes in the ATP/ADP ratio, O-2 consumption and pyridine and flavin nucleotide fluorescence preceded the beginning of the Ca2+ chan ge. These relationships are consistent with a model in which phosphory lation of glucose is the initial event which generates the signals tha t 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 functi on as the initiator of increased mitochondrial respiration.