Incretin-modulated beta cell energetics in intact islets of Langerhans

Abstract

Incretins such as glucagon-like peptide 1 (GLP-1) are released from the gut and potentiate insulin release in a glucose-dependent manner. Although this action is generally believed to hinge on cAMP and protein kinase A signaling, up-regulated beta cell intermediary metabolism may also play a role in incretin-stimulated insulin secretion. By employing recombinant probes to image ATP dynamically in situ within intact mouse and human islets, we sought to clarify the role of GLP-1-modulated energetics in beta cell function. Using these techniques, we show that GLP-1 engages a metabolically coupled subnetwork of beta cells to increase cytosolic ATP levels, an action independent of prevailing energy status. We further demonstrate that the effects of GLP-1 are accompanied by alterations in the mitochondrial inner membrane potential and, at elevated glucose concentration, depend upon GLP-1 receptor-directed calcium influx through voltage-dependent calcium channels. Lastly, and highlighting critical species differences, beta cells within mouse but not human islets respond coordinately to incretin stimulation. Together, these findings suggest that GLP-1 alters beta cell intermediary metabolism to influence ATP dynamics in a species-specific manner, and this may contribute to divergent regulation of the incretin-axis in rodents and man.

Figure 1.

The ATP/ADP-sensor Perceval reports beta cell metabolism in intact islets. A, Specific immunohistochemistry for insulin demonstrates the preferential expression of Perceval in beta cells (DAPI, blue; scale bar shown). B, As for A, but immunostaining against glucagon showing absence of the probe in alpha cells. C, Elevated glucose concentration increases Perceval fluorescence reflecting increases in cytoplasmic ATP/ADP ratio ([ATP/ADP]_cyto). D, Representative recording of a Perceval-expressing islet before and during exposure to high glucose (G17; 17 mM glucose) (n = 14 recordings from six animals) (red, raw; gray, smoothed) (scale bar, 50 μm). E, Glucose engages a subpopulation (44%) of metabolically-active beta cells, which respond with [ATP/ADP]_cyto increases. F, Dose-response graph depicting ATP-induced increases in dialyzed Perceval fluorescence, which are unaffected by the presence of increasing cAMP concentrations (0–1 μM) (sigmoidal dose-response fitted to 2–3 independent repeats).

Figure 2.

GLP-1 induces [ATP/ADP]_cyto increases in intact islets under low and high glucose conditions. A, 20 nM GLP-1 increases cytoplasmic ATP in islets perifused with 17 mM glucose (G17) (top panel; representative trace; red, raw; gray, smoothed). Wavelet analysis shows the effects of GLP-1 on the period and power (0–1 = blue-red) of ATP oscillation frequency. B, As for A, but islets exposed to 3 mM glucose (G3). C, GLP-1 engages a subpopulation of metabolically-coupled beta cells at 17 mM glucose. D, As for C but in the presence of 3 mM glucose. E, Representative images showing Perceval fluorescence in an islet before and after introduction of GLP-1 (image cropped to display a single islet; scale bar, 115 μm). F, Glucose concentration does not modulate the effects of GLP-1 on ATP oscillation frequency (NS, nonsignificant; Mann-Whitney U test on the nonbinned data, n = 13 recordings from six animals). G, Cyclosporin A (CysA) does not prevent appearance of downward deflections in [ATP/ADP]_cyto (NS, nonsignificant vs GLP-1 + CysA; Student paired t test, n = 8 recordings from four animals). H, Glucose and GLP-1 are equipotent at elevating [ATP/ADP]_cyto in islets and the incretin can elicit additional increases at high (17 mM) glucose concentration (NS, nonsignificant and **, P < .01 vs G3; one-way ANOVA followed by Bonferonni's post hoc test, n = 13–14 recordings from six animals).

Figure 3.

GLP-1 decreases mitochondrial potential and engages its cognate receptor to increase [ATP/ADP]_cyto. A, Elevated glucose (17 mM; G17) stimulates rapid and large excursions in TMRE fluorescence, indicating mitochondrial hyperpolarisation (representative trace from n = 6 recordings from four animals). B, GLP-1 under elevated glucose concentration slowly and subtly increases TMRE fluorescence, indicating minor hyperpolarizing effects on mitochondrial potential (representative trace from n = 6 recordings from four animals). C, As for B but in the presence of 3 mM glucose (representative trace from n = 7 recordings from four animals). D, TMRE responses to GLP-1 are significantly lower than those to glucose (**, P < .01 vs G17-alone, one-way ANOVA followed by Bonferonni's post hoc test). E, Representative Perceval trace showing reversible blockade of GLP-1 effects by 100 nM exendin 9-39 (Ex 9-39) (gray, raw; red, smoothed). F, Ex 9-39 abolishes GLP-1-induced [ATP/ADP]_cyto dynamics and rises (*, P < .05 vs GLP-1 + Ex 9-39, Student paired t test, n = 11 recordings). G, Forced elevations in cAMP using forskolin (FSK) lead to increases in [ATP/ADP]_cyto. H, FSK evoked ATP rises in all experiments performed (13/13). Unless otherwise stated, experiments are from islets taken from ≥ three animals.

Figure 4.

GLP-1 alters beta cell metabolism to initiate the Ca²⁺ influx required to support further [ATP/ADP]_cyto increases. A, Simultaneous recordings of Perceval (blue) and Fura-Red (red) in single beta cells reveal that GLP-1-stimulated ATP rises precede those of Ca²⁺ influx. B, Summary bar graph showing the advancement of [ATP/ADP]_cyto responses relative to those of Ca²⁺ when cells are stimulated with GLP-1 (measured period is shown with arrows on A and B) (n = 5 recordings). C, As for A, but 200 μM tolbutamide (Tolb) to stimulate large increases in Ca²⁺ and ensuing ATP consuming processes (n = 5 recordings). D, Representative traces showing effects of 10 μM forskolin (FSK) and 100 μM isobutylmethylxanthine (IBMX) on [ATP/ADP]_cyto in dissociated beta cells (n = 5 recordings). E, Extracellular Ca²⁺ chelation using EGTA suppresses ATP-responses to GLP-1 (representative trace from n = 11 recordings; red, smoothed; gray, raw). F, EGTA blocked GLP-1 actions in all experiments conducted (9/9). G, As for E, but in the presence of 10 μM of the L-type VDCC blocker verapamil. H, Verapamil (Ver) abolishes GLP-1-induced [ATP/ADP]_cyto dynamics and rises (**, P < .01 vs GLP-1 + verapamil, Student paired t test, n = 11 recordings).

Figure 5.

GLP-1-regulated [ATP/ADP]_cyto dynamics are species-specific. A, Top panel: GLP-1 induces highly synchronized [ATP/ADP]_cyto oscillations in mouse islets (representative traces from three individual beta cells). Although GLP-1 induces similar ATP rises in human islets (68), population dynamics are largely stochastic. Bottom panel: heatmap depicting min-max (0–100%) for each cell in grayscale. B, Mean percentage significantly correlated cell pairs is lower in GLP-1-treated human vs mouse islets (**, P < .01 vs mouse; Mann-Whitney U test) (n = 8 recordings from three donors and four animals). C, Representative connectivity map displaying the location (x-y) of significantly correlated cell pairs from the GLP-1-responsive beta cell population. Note the relative paucity and weakness of correlated links in human vs mouse islets (P < .05) (correlation strength is color-coded; 0 [blue] = lowest, 1 [red] = highest).

Figure 6.

Schematic of GLP-1-modulated beta cell energetics. Glycolytic metabolism of glucose stimulates insulin secretion through increases in [ATP/ADP]_cyto and cAMP, leading to opening of VDCC and Ca²⁺ influx, the latter reinforcing ATP synthesis. GLP-1 augments glucose-stimulated insulin secretion by increasing cAMP input, leading to potentiated [ATP/ADP]_cyto rises and Ca²⁺ influx.