487
Glucose causes a pronounced activation of ATP-synthase dependent respiration in pancreatic beta
cells
A. Wiederkehr;
Diabetes and Metabolism, Nestlé Institute of Health Sciences, Lausanne, Switzerland.
Background and aims: Mitochondria are essential for metabolism-secretion coupling in pancreatic beta
cells. Glucose stimulation accelerates mitochondrial oxidative metabolism and respiration to increase the
cytosolic ATP/ADP ratio, which initiates plasma membrane electrical activity and thereby insulin granule
exocytosis. Cytosolic calcium signals occurring as a result of plasma membrane depolarization are sensed
by mitochondria and serve as a potentiating signal during second phase insulin secretion. The proposed
main target of mitochondrial calcium signals are pyruvate dehydrogenase, α-ketoglutarate dehydrogenase
and isocitrate dehydrogenase. Their activation will increase TCA cycle activity and the formation of
NADH. Here the effect of beta cell calcium signaling on the regulation of glucose-induced NAD(P)H
formation and ATP-synthase dependent respiration were assessed.
Materials and methods: Glucose-induced respiration was studied in INS-1E cells or groups of intact
human islets. Mitochondrial respiration was calculated as the difference before and after addition of
rotenone (complex I inhibitor) in combination with antimycinA (complex III inhibitor). ATP-synthase
dependent respiration was determined similarly using oligomycin. Oxidative metabolism in human islets
was followed by measuring NAD(P)H autofluorescence on a two-photon confocal microscope. Cytosolic
calcium signals were analyzed at the single cell level in INS-1E and primary beta cells using the calcium
sensitive probe YC3.6.
Results: Glucose stimulates mitochondrial respiration in INS-1E and human beta cells. We find that in
insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATPsynthase independent. Following glucose stimulation this fraction of the total respiratory rate remains
constant while ATP-synthase dependent respiration is strongly induced. This activation of ATP-synthase
is almost completely suppresses when calcium signaling is prevented. Under similar conditions,
NAD(P)H levels rapidly increase in response to glucose. At this point the NAD(P)H/NAD(P) ratio is in a
new steady-state determined by on the one side enhanced oxidative metabolism and on the other reoxidation by the respiratory chain. Consistent with this view, when glucose is removed the NAD(P)H
signal rapidly decreases while inhibition of respiration further augments the NAD(P)H/NAD(P) ratio.
Interestingly, calcium signaling had no impact on the NAD(P)H steady-state signal.
Conclusion: Glucose-induced calcium signaling is required to stimulate ATP-synthase dependent
respiration in pancreatic beta cells. In contrast calcium signaling does not influence steady-state
NAD(P)H levels, which are determined mainly by the glucose concentration. The data are consistent with
the hypothesis that calcium accelerates oxidative metabolism (NADH formation) and respiration (NADH
oxidation) about equally.