Phase Analysis of Metabolic Oscillations and Membrane Potential in Pancreatic Islet β-Cells

Abstract

Metabolism in islet β-cells displays oscillations that can trigger pulses of electrical activity and insulin secretion. There has been a decades-long debate among islet biologists about whether metabolic oscillations are intrinsic or occur in response to oscillations in intracellular Ca(2+) that result from bursting electrical activity. In this article, the dynamics of oscillatory metabolism were investigated using five different optical reporters. Reporter activity was measured simultaneously with membrane potential bursting to determine the phase relationships between the metabolic oscillations and electrical activity. Our experimental findings suggest that Ca(2+) entry into β-cells stimulates the rate of mitochondrial metabolism, accounting for the depletion of glycolytic intermediates during each oscillatory burst. We also performed Ca(2+) clamp tests in which we clamped membrane potential with the KATP channel-opener diazoxide and KCl to fix Ca(2+) at an elevated level. These tests confirm that metabolic oscillations do not require Ca(2+) oscillations, but show that Ca(2+) plays a larger role in shaping metabolic oscillations than previously suspected. A dynamical picture of the mechanisms of oscillations emerged that requires the restructuring of contemporary mathematical β-cell models, including our own dual oscillator model. In the companion article, we modified our model to account for these new data.

Figure 1

Phase analysis of β-cell glycolytic oscillations. (A) Oscillations in β-cell membrane potential (Vm) stimulated by 10 mM glucose (10G) were used to define the silent phase (SP) and active phase (AP). As depicted, phase was defined as a fraction of the AP. (B) Response of FBP-sensitive PKAR FRET to glucose (0–40 mM, n = 18). (C) Example recording of Vm-FBP oscillations (n = 7). (D) The relationship between the period of Vm and FBP is plotted for individual oscillations (open circles) and islet averages (solid boxes), which were fit by linear regression. (E) The phase of each FBP peak and nadir was calculated relative to Vm as in (A) and plotted as a histogram. The charts reflect 24 APs from seven simultaneous Vm-PKAR recordings. (F) The DOM clearly predicts a different phase relationship than we observed, with the FBP and Vm being largely in phase. Equations for the model are described in the companion article (39). To see this figure in color, go online.

Figure 2

Phase analysis of β-cell mitochondrial flavin⁻¹ oscillations. (A) Simultaneous recording of islet NAD(P)H and flavin⁻¹ oscillations (n = 20). (B–D) Example recordings of Vm-flavin⁻¹ oscillations in 10 mM glucose (n = 15). (E) The phase of each flavin⁻¹ nadir and largest peak was calculated relative to Vm as shown in Fig. 1A and plotted as a histogram. The chart reflects 40 APs from 15 simultaneous Vm-flavin⁻¹ recordings. (F) The relationship between Vm period and flavin⁻¹ period is plotted for individual oscillations (open circles) and islet averages (solid boxes). Islet averages were then fit by linear regression. To see this figure in color, go online.

Figure 3

Relationship between islet NAD(P)H and mitochondrial membrane potential (ΔΨm). Mitochondrial membrane potential is plotted as Rhodamine-1,2,3 fluorescence (n = 30 islets from three animals). Note that a decrease in fluorescence corresponds to ΔΨm hyperpolarization and increased driving force for ATP production. To see this figure in color, go online.

Figure 4

Phase analysis of β-cell ATP/ADP oscillations. (A) Example recording of Vm-ATP/ADP oscillations in 10 mM glucose (n = 13). (B) The phase of each ATP/ADP peak and nadir was calculated relative to Vm as shown in Fig. 1 A and plotted as a histogram. The charts reflect 40 APs from 10 simultaneous Vm-ATP/ADP recordings. (C) The relationships between Vm period and ATP/ADP period are plotted for individual oscillations (open circles) and islet averages (solid boxes). Islet averages were then fit by linear regression. (D) Example recording of ATP/ADP and cytosolic Ca²⁺ oscillations in 10 mM glucose (n = 10). To see this figure in color, go online.

Figure 5

Effects of steady-state Ca²⁺ on the β-cell triggering pathway. The effects of lowering islet Ca²⁺ with diazoxide (Dz, 200 μM) and raising Ca²⁺ with Dz/KCl ([K⁺]_o = 30 mM) were assessed by simultaneous measurements of Vm and intracellular Ca²⁺ (n = 4) (A), FBP (n = 4) (B), flavin⁻¹ (n = 5) (C), or cytosolic Ca²⁺ and ATP/ADP (n = 45) (D). To see this figure in color, go online.

Figure 6

Glycolytic oscillations can occur without Ca²⁺ oscillations. After suppression of FBP oscillations with Dz, stepwise elevation of [K⁺]_o from 15 to 30 mM restored oscillations in two of 25 islets (8%) in five experiments (A). In the majority of islets, oscillations were not restored (B). To see this figure in color, go online.