Abstract
Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed.
Highlights
Stimulation of insulin secretion in the pancreatic 0- cells has not yetbeen determined
The results demonstrated that the steady-state free Ca2+concentration varied inversely with the ATP/ ADP ratio andorthophosphate (Pi)levels
Searching for signals that could be common to themetabolic pathways of various stimulatory fuels, we focused our attention on shared intracellular cofactors or nucleotides as couof the Ca2+set point by the ATP/ADP ratio was ob- pling factors linking fuel metabolism, ionic alterations, and served at ratiosbetween 5 and 50 and at Piconcentra- insulin secretion
Summary
RINm5F insulinoma cells were incubated at 30 “C in 2.5 mlof a buffer (pH 7.3) containing the following components: 100 mM KCl, 22 mM NaCl, 5 mM KHC03, 20 mM HEPES, 1 mM MgCl,, 4 mM KH,PO,, 4 mM MgATP, 12 mM creatine plus creatine phosphate, and 50 pg/ml creatine phosphokinase. The results indicate that there is an inverse correlation be- cytochrome BCI complex)and oligomycin (to block mitochontween both the ATP/ADP ratio and phosphate levels (but drial ATPase activity).As shown in Fig. 3B a similar inverse not the phosphorylation potential) and theCa2+steady state relationship between the Ca2+set point and the ATP/ADP maintained by the ER compartment. They indicate that ratio persisted underthese conditions.
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