Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca2+ Mobilization From Acidic Stores in Pancreatic α-Cells

Abstract

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of β-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca2+ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca2+]i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca2+ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca2+]i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca2+ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca2+]i Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that β-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca2+]i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca2+ release from the acidic stores and further amplified by Ca2+-induced Ca2+ release from the sarco/endoplasmic reticulum.

Conflict of interest statement

Conflict of interest

Authors declare no conflicts of interest.

© 2018 by the American Diabetes Association.

Figures

Figure 1. The stimulatory effect of adrenaline on glucagon secretion is mediated by selective elevation of [Ca2+]i in pancreatic α-cells

(a) Glucagon secreted from isolated NMRI mouse islets in response to 3mM glucose with/without 5μM adrenaline. #p<0.05 vs the effect of 3 mM glucose alone. (b) Variance of the Fluo4 intensity when the islet was perifused with 3mM glucose ± 5μM adrenaline or 20mM glucose (as indicated). The brighter cells are those in which [Ca2+]i oscillates. The arrow indicates a cell that started spiking after adrenaline had been applied. (c-d) Typical single α-cell responses to application of 1mM glutamate and 5μM adrenaline recorded in mouse (c; n=29) and human (d; n=55) islets, at 3mM glucose. (e) Representative [Ca2+]i timecourse in the populations of α- (n=21) and non-α-cells (mostly, β-cells, n=75), differentiated by the response to glutamate. The difference in magnitude of the glutamate and the adrenaline effects was not a consistent finding. (f) [Ca2+]i changes in α-cells quantified as pAUC at 3mM glucose ± glutamate or adrenaline in mouse (NMRI, C57Bl/6N) and human islets. p<0.05 vs the respective effect observed in NMRI mice (*) or the effect of the basal (3 mM glucose) in the same recording (#).

Figure 2. Adrenaline’s effect in pancreatic α-cells depends on Ca2+ influx through L-type Ca2+ channels and is mediated by β-adrenergic signaling

(a) Glucagon secreted from isolated NMRI mouse islets in response to 3 mM glucose with(out) 5μM adrenaline ± isradipine or ω-agatoxin (as indicated). p<0.05 vs the effect of adrenaline + 3mM glucose (*) or vs the basal (3mM glucose + respective antagonist) group. (b) [Ca2+]i upon adrenergic (isoprenaline (n=27), or noradrenaline (n=19)) stimulation alone (n=29) or with (as indicated) isradipine (n=17, preincubated, n=29, acute), ω-agatoxin (n=13), propranolol (n=11), prazosin (n=49), diazoxide (n=20), EGTA (n=84). p<0.05 vs. the effect of adrenaline alone (*) or vs the basal (3 mM glucose+respective (ant)agonist) of the same recording. (c) Effect of adrenaline on α-cell action potential firing (representative of 11 experiments). Examples of action potentials recorded in the absence and presence of adrenaline (taken from the recording above as indicated) are shown.

Figure 3. Adrenaline mediates its effects via elevation of [cAMP]i

(a) Glucagon secretion from isolated NMRI mouse islets in response to 3mM glucose. Adrenaline, myr-PKI or ESI-05 was added as indicated. p<0.05 vs the basal (3 mM glucose + respective antagonist) (#), vs the effect of 3 mM glucose + adrenaline (*) or vs the effect of 3mM glucose + adrenaline in the presence of ESI-05 (¶). (b) [Ca2+]i upon application of (as indicated) adrenaline ± myr-PKI (n=32) or ESI-05 (n=12). p<0.05 vs the basal of the same recording (#) or vs the effect of 3 mM glucose + adrenaline (*). (c) Representative recording of the depolarization-induced increases in plasma membrane electrical capacitance. (d) Exocytosis in α-cells. The pipette solution contained (as indicated) either no cAMP (n=7) or 0.1mM cAMP (n=11) and PKA (1μM PKI, n=32) or EPAC2 (25μM ESI-05, n=11). p<0.05 vs the effect of addition of cAMP into the pipette solution (*) and vs the control (cAMP-free) condition (#). (e) The effect of adrenaline on PKA activity in α- (n=22) and β-cells (n=85) within pancreatic islet cells isolated from Glu-RFP mice imaged using AKAR-3 sensor on LSM510 confocal microscope (see also Figure S3). The PKA activity is expressed as a change of the FRET ratio of the AKAR-3 sensor. (f) Comparison of the effects of 5μM adrenaline and 1nM-10μM forskolin on PKA activity of pancreatic islet cells (n=870). The excerpt (below, n=62) represents the data from cells activated by adrenaline (α-cells, see Figure 3e). Note the higher sensitivity of α-cells to forskolin: EC50=187±50nM (n=24) and 383±7 nM (n=807) for α- and β-cells, respectively. (g) Representative concentration-PKA activation dependence of [Ca2+]i on forskolin in α-cells measured using Fluo4. (h) Forskolin concentration-activation curves for PKA (solid line, n=98) and [Ca2+]i (dashed line, n=20). The effect of 5μM adrenaline on PKA measured on the same cells is mapped on to the curves as a shaded area (4.1±0.8μM forskolin).

Figure 4. Adrenaline-induced glucagon secretion involves intracellular Ca2+ release

(a) Glucagon secreted from isolated NMRI mouse islets at 3mM with or without adrenaline and ryanodine, xestospongin C or thapsigargin as indicated. p<0.05 vs the basal (#) or vs the effect of glucose + adrenaline (*). (b) Representative recording of the effect of ryanodine on adrenaline-induced [Ca2+]i increases visualized using Fluo4FF. The control (dashed) is superimposed with the experimental trace. (c) [Ca2+]i upon adrenaline stimulation alone (n=23) or in the presence of ryanodine (n=22), xestospongin C (n=30) or CPA (n=14), as measured using Fluo4FF. (d) [Ca2+]i upon adrenaline stimulation alone or in the presence of Ned-19 (n=28), bafilomycin (n=36), myr-PKI and ESI-05 (n=31) or myr-PKI + ESI-05 + Ned-19 (n=21). p<0.05 vs the basal (3 mM glucose + antagonist) of the same recording (#) or vs the effect of 3 mM glucose + adrenaline (*).

Figure 5. CD38 and TPC2 but not TPC1 mediate the adrenaline response in α-cells

(a) Effect of adrenaline on [Ca2+]i in α-cells within islets isolated from TPC1-/- or TPC2-/- mice as indicated. (b) [Ca2+]i upon adrenaline stimulation in wild-type, TPC1-/- (n=84), TPC2-/- (n=27) or CD38-/- (n=153) mouse α-cells. p<0.05 vs basal (3 mM glucose) of the same recording (#) or vs the effect of 3 mM glucose + adrenaline (*). (c) Glucagon secretion from isolated mouse islets in response to low glucose (3mM) or adrenaline in the absence/presence of Ned-19 or ryanodine, measured in wild-type C57Bl/6n and TPC1-/-, TPC2-/- and CD38-/- mice. p<0.05 vs the basal (3 mM glucose) (#), vs the effect of 3 mM glucose + adrenaline within the same genotype (*) or vs the effect of 3mM glucose + adrenaline in wild-type animals (§).

Figure 6. The adrenaline effect is attenuated by chronic hyperglycemia

(a) Typical [Ca2+]i responses to 5μM adrenaline recorded in mouse islet α-cells precultured at 11 mM (n=102) or 30 mM (n=146) glucose for 48 hours. (b) Quantification of the data presented in (a). p<0.05) vs the basal (3 mM glucose) of the same recording (#) or vs the effect of 3 mM glucose + adrenaline in islets pre-cultured in 11 mM glucose (¶). (c) cAMP responses to adrenaline in pancreatic islet α-cells pre-cultured at 11 (n=164) or 30mM (n=212) glucose. (d) PKA responses to adrenaline in pancreatic islet α-cells pre-cultured at 11 (n=1277) or 30 mM (n=1323) glucose. (e) Glucagon secretion from pancreatic islets pre-cultured at 11 (n=10) or 30 mM (n=10) glucose. p<0.05) vs the basal (3 mM glucose) condition (#) or vs the respective effect in islets precultured in 11 mM glucose (¶).

Figure 7. Model of adrenaline-induced glucagon secretion. See main text for details.

AC, adenylyl cyclase; Cav1.x, L-type voltage-gated Ca2+ channel; Gs, G-protein, s α-subunit. See main text for description.