Glucose-stimulated single pancreatic islets sustain increased cytosolic ATP levels during initial Ca2+ influx and subsequent Ca2+ oscillations

Abstract

In pancreatic islets, insulin secretion occurs via synchronous elevation of Ca(2+) levels throughout the islets during high glucose conditions. This Ca(2+) elevation has two phases: a quick increase, observed after the glucose stimulus, followed by prolonged oscillations. In these processes, the elevation of intracellular ATP levels generated from glucose is assumed to inhibit ATP-sensitive K(+) channels, leading to the depolarization of membranes, which in turn induces Ca(2+) elevation in the islets. However, little is known about the dynamics of intracellular ATP levels and their correlation with Ca(2+) levels in the islets in response to changing glucose levels. In this study, a genetically encoded fluorescent biosensor for ATP and a fluorescent Ca(2+) dye were employed to simultaneously monitor the dynamics of intracellular ATP and Ca(2+) levels, respectively, inside single isolated islets. We observed rapid increases in cytosolic and mitochondrial ATP levels after stimulation with glucose, as well as with methyl pyruvate or leucine/glutamine. High ATP levels were sustained as long as high glucose levels persisted. Inhibition of ATP production suppressed the initial Ca(2+) increase, suggesting that enhanced energy metabolism triggers the initial phase of Ca(2+) influx. On the other hand, cytosolic ATP levels did not fluctuate significantly with the Ca(2+) level in the subsequent oscillation phases. Importantly, Ca(2+) oscillations stopped immediately before ATP levels decreased significantly. These results might explain how food or glucose intake evokes insulin secretion and how the resulting decrease in plasma glucose levels leads to cessation of secretion.

Keywords: ATP; Calcium; Energy Metabolism; Imaging; Insulin; Pancreatic Islets.

FIGURE 1.

Validation of GO-ATeam in insulin-secreting cells. Time course of the fluorescence emission ratio (OFP/GFP) in permeabilized MIN6 cells expressing GO-ATeam1 biosensors in the cytosol. A, after permeabilization, cells were perfused with intracellular-like medium including different concentrations (2–10 mm) of MgATP (n = 9). B, after permeabilization, cells were alternately perfused with intracellular-like medium including 7 or 8 mm MgATP (n = 10). C, average time course of the fluorescence emission ratio (OFP/GFP) of MIN6 cells expressing GO-ATeam1 biosensors in the cytosol. OFP/GFP ratios were monitored when medium glucose was increased from 2.8 to 25 mm (n = 22). Cells that did not exhibit clear increases in response to changing glucose levels were excluded from the data analysis. The error bars indicate S.D.

FIGURE 2.

Glucose stimulation induces a rapid increase in cytosolic and mitochondrial ATP levels in single isolated mouse islets. A and B, time course of the fluorescence emission ratio (OFP/GFP) of isolated islets expressing GO-ATeam biosensors in the cytosol. OFP/GFP ratios of GO-ATeam1 (A) or GO-ATeam3 (B) were monitored when medium glucose was increased from 2.8 to 25 mm (n = 13 and 4, respectively). C, time course of the fluorescence emission ratio (YFP/CFP) of isolated islets expressing ATeam1.03 (AT1.03) biosensors in the cytosol. YFP/CFP ratios were monitored when medium glucose was increased from 2.8 to 25 mm (n = 4). D, the average time course of OFP/GFP ratios of isolated islets expressing GO-ATeam1. OFP/GFP ratios were monitored in the same islets when glucose was increased from 2.8 to 8.3, 16.7, and 25 mm (n = 10). The error bars indicate S.D. E, the amplitudes of OFP/GFP ratio changes were quantified in various glucose conditions shown in D. The error bars indicate S.D., and asterisks indicate significant differences. *, p < 0.05; **, p < 0.0001. F, time course of OFP/GFP ratios of isolated islets stimulated with various levels of glucose. Glucose levels were increased in a stepwise manner from 2.8 to 25 mm (n = 8). G, time course of the fluorescence emission ratio (OFP/GFP) of single cells isolated from islets expressing GO-ATeam1. The OFP/GFP ratio was monitored when glucose in the medium was increased from 2.8 to 25 mm, followed by 5 μg/ml oligomycin A addition (n = 11). H and I, time course of the fluorescence emission ratio (OFP/GFP) of isolated islets expressing GO-ATeam biosensors in the mitochondrial matrix. OFP/GFP ratios of mitGO-ATeam1 (H) or mitGO-ATeam3 (I) were monitored (n = 7 and 4, respectively).

FIGURE 3.

Cytosolic ATP level responds more rapidly to an increase than to a decrease in glucose concentration in the medium. A, dynamics of the OFP/GFP ratio in isolated islets expressing GO-ATeam1 in response to alternating glucose concentrations between 2.8 and 25 mm in the medium (n = 9). B, the average rates of change of OFP/GFP ratios upon glucose increase or decrease. The values are calculated from experiments as in A. C, times required for the OFP/GFP ratio to reach 50% of the maximum value after changes in glucose concentration. The values are calculated from experiments as in A. The error bars indicate S.D., and asterisks indicate significant differences (p < 0.0001).

FIGURE 4.

Both iodoacetate and oligomycin A rapidly lower [ATP]_c in pancreatic islets. A, time course of [ATP]_c in a single islet treated with 1 mm iodoacetate (n = 5). B, time course of [ATP]_c in a single islet treated with 5 μg/ml oligomycin A (n = 5). Islets were incubated in KRH medium containing 25 mm glucose.

FIGURE 5.

The increase in [Ca²⁺]_c follows that of [ATP]_c in glucose-stimulated isolated islets. A, co-imaging of [ATP]_c and [Ca²⁺]_c in single isolated glucose-stimulated islets using GO-ATeam1 and fura-2. Glucose in the medium was increased from 2.8 to 25 mm. Pseudocolored ratiometic images of GO-ATeam1 (OFP/GFP ratio, referred to as ATP) and fura-2 (340ex/380ex ratio, referred to as Ca²⁺) are shown. The italicized letters correspond to those in B. B, representative time courses of [ATP]_c and [Ca²⁺]_c in glucose-stimulated islets (n = 19). [ATP]_c and [Ca²⁺]_c within the region of interest (white circular area) of the islet represented in A are shown. C and D, representative time courses of [ATP]_c and [Ca²⁺]_c in glucose-stimulated (C) or tolbutamide-stimulated (D) islets pretreated with 1 mm iodoacetate (n = 6 for both C and D, respectively). E and F, representative time courses of [ATP]_c and [Ca²⁺]_c in glucose-stimulated (E) or tolbutamide-stimulated (F) islets pretreated with 1 μg/ml oligomycin A (n = 6 for both E and F, respectively). The black lines represent [ATP]_c, and the red lines represent [Ca²⁺]_c.

FIGURE 6.

Methyl pyruvate induces increases of intracellular ATP levels prior to Ca²⁺ influx. Results shown are for [ATP]_c, [ATP]_m, and [Ca²⁺]_c in single isolated islets stimulated with MP. Islets were incubated in KRH medium without glucose for 40 min. A, representative time course of [ATP]_m and [Ca²⁺]_c in single isolated islets stimulated with 20 mm MP (n = 5). B, representative time course of [ATP]_c and [Ca²⁺]_c in single isolated islets stimulated with 20 mm MP (n = 7). C, representative time courses of [ATP]_c and [Ca²⁺]_c in single isolated islets stimulated with 1 μg/ml oligomycin A and 20 mm MP (n = 6). The black lines represent [ATP]_m (A) or [ATP]_c (B and C), and the red lines represent [Ca²⁺]_c.

FIGURE 7.

Treatment with both leucine and glutamine leads to increases in intracellular ATP levels prior to Ca²⁺ influx. [ATP]_c, [ATP]_m, and [Ca²⁺]_c were monitored in single isolated islets treated with leucine and/or glutamine. Islets were incubated in KRH medium containing 2.8 mm glucose. A and B, dynamics of intracellular ATP and Ca²⁺ levels in leucine-stimulated single islets. [ATP]_m (A) and [ATP]_c (B) were monitored along with [Ca²⁺]_c in single isolated islets treated with 10 mm leucine (n = 5 and 4, respectively). C and D, dynamics of intracellular ATP and Ca²⁺ levels in glutamine-stimulated single islets. [ATP]_m (C) and [ATP]_c (D) were monitored along with [Ca²⁺]_c in single isolated islets treated with 10 mm glutamine (n = 3 and 5, respectively). E and F, dynamics of intracellular ATP and Ca²⁺ levels in leucine/glutamine-stimulated single islets. [ATP]_m (E) and [ATP]_c (F) were monitored along with [Ca²⁺]_c in single isolated islets treated with 10 mm leucine in the presence of 10 mm glutamine (n = 5 for both E and F, respectively). G, representative time courses of [ATP]_c and [Ca²⁺]_c in single isolated islets stimulated with 1 μg/ml oligomycin A and 10 mm leucine in the presence of 10 mm glutamine (n = 6). Islets were pretreated with glutamine for 20 min (E–G) before initiating the imaging experiments. The black lines represent [ATP]_m (A, C, and E) or [ATP]_c (B, D, F, and G), and the red lines represent [Ca²⁺]_c.

FIGURE 8.

Correlation between [ATP]_c and [Ca²⁺]_c when [Ca²⁺]_c was oscillated under high glucose conditions. A, representative time courses of [ATP]_c and [Ca²⁺]_c in glucose-stimulated islets showing oscillations in [Ca²⁺]_c (n = 15). Data for the four plots shown in this figure were from different islets. Islets were stimulated by increasing the glucose concentrations in the medium from 2.8 to 25 mm. B, cross-correlation analysis of the dynamics of [ATP]_c and [Ca²⁺]_c. Cross-correlation analysis was performed between the dynamics of [ATP]_c and [Ca²⁺]_c in the rectangle shown in A. The trace shows the one-dimensional cross-correlation, with the time difference (t) of the correlation on the x axis and the numerically expressed cross-correlation amplitude on the y axis. C, dynamics of [ATP]_c during Ca²⁺ oscillations induced by high glucose levels and further augmentation of glucose in the medium. [ATP]_c and [Ca²⁺]_c in single islets were monitored after stimulation with 25 mm and further with 42 mm glucose (n = 6). D, representative time course of [ATP]_c and [Ca²⁺]_c in an islet stimulated with various levels of glucose. Glucose levels were increased in a stepwise manner from 2.8 to 25 mm (n = 12). The black lines represent [ATP]_c, and the red lines represent [Ca²⁺]_c.

FIGURE 9.

Dynamics of [ATP]_c and [Ca²⁺]_c when glucose-induced [Ca²⁺]_c oscillations were stopped. A, representative time course of [ATP]_c and [Ca²⁺]_c in islets showing [Ca²⁺]_c oscillations, treated with 4 μm carbonylcyanide 3-chlorophenylhydrazone (CCCP, n = 5). Islets were incubated in KRH medium containing 25 mm glucose. B, representative time course of [ATP]_c and [Ca²⁺]_c in islets upon reduction of the glucose concentration (n = 5). The black lines represent [ATP]_c, and the red lines represent [Ca²⁺]_c.

FIGURE 10.

Dependence of glucose-induced intracellular ATP elevation on intracellular Ca²⁺. A–D, the effect of Ca²⁺ depletion from the medium. [ATP]_m (A and B) or [ATP]_c (C and D) was monitored in isolated islets when the glucose level was elevated from 2.8 to 20 mm (n = 5 for both A and C, n = 6 for both B and D, respectively). Islets were pretreated with Ca²⁺-depleted KRH medium (A and C) or normal Ca²⁺-containing KRH medium (B and D) for 40 min. E and F, the effect of intracellular Ca²⁺ depletion. [ATP]_m (E) or [ATP]_c (F) was monitored in isolated islets when the glucose level was elevated from 2.8 to 20 mm (n = 5 for both E and F, respectively). Islets were pretreated with 5 μm BAPTA-AM for 20 min. G, depletion of intracellular Ca²⁺ decreases basal [ATP]_m. Time course of [ATP]_m in islets treated with 5 μm BAPTA-AM is shown. Islets were incubated in KRH medium containing 2.8 mm glucose (n = 5).