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. 2012 May 4;287(19):15706-17.
doi: 10.1074/jbc.M111.314567. Epub 2012 Mar 14.

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

Isabel Goehring  1 , Akos A Gerencser, Sara Schmidt, Martin D Brand, Hindrik Mulder, David G Nicholls
Affiliations

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

Isabel Goehring et al. J Biol Chem. .

Abstract

Oscillations in plasma membrane potential play a central role in glucose-induced insulin secretion from pancreatic β-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (Δψ(p)) indicator in combination with indicators of cytoplasmic free Ca(2+) ([Ca(2+)](c)), mitochondrial membrane potential (Δψ(m)), matrix ATP concentration, and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS-1 832/13 β-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca(2+) concentration over the same concentration range required for insulin release, although considerable cell-to-cell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8 mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters (Δψ(p) and [Ca(2+)](c), Δψ(p) and NAD(P)H, matrix ATP and [Ca(2+)](c), and Δψ(m) and [Ca(2+)](c)) were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in Δψ(p) are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded, an autonomous Δψ(p) oscillatory mechanism is initiated.

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Figures

FIGURE 1.
FIGURE 1.
Δψp responses to 14 mm glucose. INS-1 832/13 cells were preincubated in buffer B in the presence of 2.8 mm glucose as described under “Experimental Procedures.” Where indicated, an additional 14 mm glucose was added, followed by oligomycin to inhibit the ATP synthase. Finally sufficient high K+ medium was substituted to increase the [K+] to 25 mm to calibrate the response. Changes in Δψp were monitored with PMPI, and the traces were normalized to equalize the change in fluorescence from the initial to final values in the experiment. The average response of the entire field is shown together with representative responses of bursting and non-bursting single cells.
FIGURE 2.
FIGURE 2.
Glucose concentration-dependent responses. Cells were preincubated in buffer A in the presence of 2.8 mm glucose and transferred to glucose-free medium immediately before imaging. Plasma membrane potential (Δψp) and cytoplasmic free Ca2+ ([Ca2+]c) were monitored in parallel. Where indicated, varying concentrations of glucose were added, followed by oligomycin. The average Δψp response of the entire field (Δψp field) and of a selected cell (Δψp cell) is shown. In parallel, changes in [Ca2+]c in the same cell are reported. The Δψp traces are normalized to a KCl calibration (see “Experimental Procedures”).
FIGURE 3.
FIGURE 3.
Heterogeneous Δψp responses of individual cells to glucose. Cells were preincubated in buffer A in the presence of 2.8 mm glucose and transferred to glucose-free medium immediately before imaging. Where indicated, 8 mm glucose (glu), 640 ng/ml oligomycin (oligo) were added. KCl was added to 25 mm. Representative Δψp and [Ca2+]c responses of three individual cells are shown following addition of 8 mm glucose. Cell a had a long latent period before initiating Δψp and [Ca2+]c oscillations. Cell b showed an initial oscillation followed by a latent period. Cell c was quiescent after the initial oscillation until oligomycin was added.
FIGURE 4.
FIGURE 4.
Pyruvate-induced Δψp oscillations in the presence and absence of 2.8 mm glucose. a, INS-1 832/13 cells were preincubated in buffer A in the presence of 2.8 mm glucose. Where indicated, 10 mm Na-pyruvate was added, followed by 640 ng/ml oligomycin (oligo) and KCl to 25 mm. b, cells were preincubated in buffer B in the absence of exogenous substrate for 2 h prior to imaging.
FIGURE 5.
FIGURE 5.
Insulin release in static 1-h incubations of INS-1 832/13 cells in the presence of the indicated concentrations of glucose or pyruvate relative to release in the absence of exogenous substrate. Data are mean ± S.E. (n = 3).
FIGURE 6.
FIGURE 6.
Mitochondrial hyperpolarization induced by glucose or pyruvate. INS-1 832/13 cells were preincubated in buffer A in the presence of 2.8 mm glucose and 100 nm TMRM. Glucose-free medium with TMRM was substituted immediately before imaging. Field-average TMRM fluorescence is shown for 140 s following addition of the indicated concentration of glucose or pyruvate. Each division corresponds to a 10% decrease in signal. Because the experiment is performed in quench mode, a decrease in TMRM fluorescence corresponds to a mitochondrial hyperpolarization. The values in brackets are the estimated extents of hyperpolarization (in mV) calculated as described in the supplemental material. Note the rapid hyperpolarization induced by 9 mm pyruvate. Changes in Δψp do not affect the signal over this brief time period (21).
FIGURE 7.
FIGURE 7.
Changes in Δψm associated with a [Ca2+]c spike in the presence of pyruvate. INS-1 832/13 cells were preincubated in buffer B in the presence of 2.8 mm glucose 100 nm TMRM (a) and 2 μm fura-2 (b, fura-2 340/380 fluorescence ratio is shown in pseudocolor). c, at the end of the recording, cells were stained with the nuclear marker DRAQ5. d, overlay image of TMRM (green), fura-2 (blue), and DRAQ5 (red) fluorescence micrographs. e, individual cells were identified by segmentation of fura-2 images using DRAQ5 as seed. f, mitochondrion-free (nuclear) areas were determined within each cell with further image segmentation. g and h, mean responses synchronized to [Ca2+]c spikes monitored with fura-2 (thin lines) and corresponding mean changes in single cell nuclear TMRM fluorescence (heavy lines) in the presence of 0.4 mm and 10 mm pyruvate in 2.8 and 0 mm glucose, respectively. Data points are mean ± S.E. of three experiments, compiled from total 516 and 564 oscillations for g and h, respectively. The mV scale bar represents the Δψm change approximated by modeling (supplemental Fig. S3).
FIGURE 8.
FIGURE 8.
Temporal correlation between plasma membrane depolarization and NAD(P)H autofluorescence. INS-1 832/13 cells were preincubated in buffer B in the presence of 2.8 mm glucose and PMPI. NAD(P)H autofluorescence (a) and PMPI (b) intensities were detected on single cell basis by staining cultures by DRAQ5 (c) at the end of each recording and segmenting the PMPI images (d). e, mean responses synchronized to PMPI spikes in the presence of 10 mm pyruvate. PMPI signal (inverted, thin line) is reported as percentage change, and the NAD(P)H autofluorescence (heavy line) is reported as a percentage of the maximal span (FCCP to rotenone, see “Experimental Procedures”). Data points are mean ± S.E. of three experiments, compiled from a total of 800 oscillations.
FIGURE 9.
FIGURE 9.
Matrix ATP concentration in INS-1 832/13 cells as a function of substrate concentration and during [Ca2+]c spikes in the presence of 0.4 mm pyruvate. Cells transfected with matrix-targeted ATeam AT1.03 (a) were loaded with Fura Red (b) during preincubation in the presence of 2.8 mm glucose. Spectrally unmixed images are shown. At the end of each recording nuclei were stained by Hoechst 33342 (c), and the ATeam fluorescence images were segmented (d) from these seeds to identify individual cells. e and f, to evaluate [ATP] and [Ca2+]c, 542/483 emission and 438/500 excitation ratios were calculated, respectively, and are shown as pseudocolor scaled images. g, the response of matrix targeted AT1.03 to pyruvate was studied in cells not loaded with Fura Red. Where indicated pyruvate (P) was added to give final concentrations of 0.1, 0.2, 0.4, 0.6, and 1 mm, followed by glucose to 16 mm and 1 μm oligomycin (oligo) plus 1 mm iodoacate (IAA). ATP concentrations were calculated using the published data for the affinity for AT1.03 (20). Data points are mean ± S.E. of four experiments. h, mean responses synchronized to [Ca2+]c spikes monitored with Fura Red (thin line) and corresponding mean changes in [ATP] (heavy line) in cells exposed to 0.4 mm pyruvate. Data points are mean ± S.E. of four experiments, compiled from a total of 130 oscillations.
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