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

Abstract

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 approximately 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 beta-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of alpha-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 alpha-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 02 oscillations were also not eliminated by the addition of relatively high concentrations of pyruvate or alpha-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 02 consumption and also prevented 02 oscillations. These results suggest that the changes in respiration reflected in the 02 oscillations occur in response to the oscillations in the ATP/ADP ratio or ADP concentration and that this parameter is a primary regulator of 02 consumption in the pancreatic beta-cell.

Are Metabolic Oscillations Responsible for Normal Oscillatory Insulin Secretion?

“…We have recently demonstrated oscillations in the ATP/ADP ratio and glucose-6-phosphate in a suspension of p-cells synchronized by clonidine treatment (51). Oscilla- tory oxygen consumption (12) also suggests oscillations in ADP or the ATP/ADP ratio (35), as do oscillations in the activity of K^p channels (52,53). The most likely mechanism for these oscillations is spontaneous glycolytic oscillations, a well-established phenomenon in other cell systems, as described above.…”

“…Also present are C-and L-type isoforms of PFK, which are not strongly activated by F16BP under physiological conditions and should not promote oscillatory behavior; however, the F16BP-sensitive activity characteristic of PFK-M appears to dominate. NADH oscillations in glycolyzing islet extracts are also indicative of the capacity for oscillatory glycolysis (35). The importance of PFK-M to stimulus-secretion coupling in the (3-cell is suggested by recent studies of humans with a genetic deficiency of this isoform, in whom the ability of intravenous glucose to stimulate insulin secretion was greatly impaired (36).…”

“…The most likely mechanism for these oscillations is spontaneous glycolytic oscillations, a well-established phenomenon in other cell systems, as described above. Furthermore, we have demonstrated that incubation of permeabilized clonal 3-cells in oscillatory glycolyzing muscle cytosol induces oscillations in oxygen consumption and Ca 2+ handling (20,35). A clear distinction should be made between spontaneous oscillations of a biochemical system and secondary oscillations due to an oscillatory input.…”

Plasma Membrane Potential Oscillations in Insulin Secreting Ins-1 832/13 Cells Do Not Require Glycolysis and Are Not Initiated by Fluctuations in Mitochondrial Bioenergetics

“…In intact islets and dissociated ␤-cells, it has been suggested that glycolytic fluctuations, perhaps controlled by phosphofructokinase, may be responsible (9,10). These are associated with oscillations of respiration (11), nicotinamide nucleotide reduction state (12), mitochondrial membrane potential (13), cytoplasmic ATP/ ADP ratios (9,14), and consequent activation and inactivation of the plasma membrane K ATP channel.…”

Mathematical modeling demonstrates how multiple slow processes can provide adjustable control of islet bursting

“…concentration. [10][11][12][13][14] Thus, the contribution that the K(ATP) current makes to bursting is determined by the contribution that changes in the variable a make. Ca 2+ acts directly to activate the SK4 channels, so SK4 conductance reflects the Ca 2+ concentration in the cytosol, which in part reflects slow changes in the ER Ca 2+ concentration.…”