GLP-1 inhibits and adrenaline stimulates glucagon release by differential modulation of N- and L-type Ca2+ channel-dependent exocytosis

Abstract

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).

Figure 1

Divergent effects of cAMP-increasing agents on glucagon secretion and involvement of PKA. (A) Glucagon secretion measured from isolated mouse islets in 0 mM glucose (Ctrl) and in the presence of 100 nM GLP-1, 100 nM GIP or 5 μM adrenaline (Adr). ***p<0.001 vs. ctrl; (B) As in A, but in the presence of 10 μM of the PKA-inhibitor 8-Br-Rp-cAMPS as indicated. ††p<0.01 vs. Ctrl; ‡p<0.05, ‡‡‡p<0.001 for comparison with corresponding values in A. Data have been normalized to control (10.4±0.5 pg/islet/h; n=8-16). (C) Glucagon secretion measured in the absence (◇) and presence (■) of 100 nM GLP-1 at different glucose concentrations (1-20 mM). **p<0.01 and ***p<0.001 for effect of GLP-1 compared at the respective glucose concentrations. Data have been normalized to control (1 mM glucose; 30.4±1.5 pg/islet/h; n=8). (D) Glucagon secretion measured at 3 mM glucose in the absence and presence of 100 nM GLP-1 with or without addition of adrenaline (Adr, 5 μM). Data have been normalized to value at 1 mM (in C; 30.4±1.5 pg/islet/h; n=8). *p<0.05, ***p<0.001 vs control and †††p<0.001 vs. GLP-1. (E) Effects of 10 nM GLP-1 in the absence and presence of 100 nM of the SSTR2 antagonist CYN154806 as indicated. Glucose was presented at 1 mM. Data have been normalized to control (2.1±0.1 pg/islet/h, n=7). **p<0.01 and ***p<0.001 vs control and ††p<0.01 vs. CYN154806 alone. (F) Expression of GLP-1 (Glp1r), GIP (Gipr) and β1 and β2-adrenergic receptors (Adrb1 and Adrb2) in mouse β-cells. (G) Same as in F but using mouse α-cells. Data have been normalized to Glp1r expression in mouse β-cells. Note use of different ordinate scales in F-G. (H) Fraction GLP-1R-positive cells of insulin- (β-cells) and glucagon-positive (α-cells). (I) Glucagon secretion at 1 mM glucose (Ctrl) and in the presence of 1 μM of exendin-(9-39) (Ex 9-39) and/or 100 nM GLP-1 as indicated. Data have been normalized to control (9.3±0.3 pg/islet/h; n=11-12). ***p<0.001 vs control; †††p<0.001 vs GLP-1 alone.

Figure 2

Concentration-dependent effects of forskolin and adrenaline on glucagon secretion and cAMP production. (A) Effects of increasing concentrations of forskolin (0-10 μM) on glucagon secretion at 0 mM glucose. Data have been normalized to control (13.0±0.9 pg/islet/h; n=8). ***p<0.001 vs. rate of secretion in the absence of forskolin. (B) Glucagon secretion at 1 mM glucose (Ctrl) with or without addition of 3 nM or 10 μM forskolin (FSK) in the absence (left) and presence (right) of 10 μM 8-Br-Rp-cAMPS. Data have been normalized to control (7.1±0.3 pg/islet/h; n=6-12).*p<0.05 and ***p<0.001 vs. Ctrl; ††p<0.01 and ‡‡‡p<0.001 vs Ctrl in the presence of 8-Br-Rp-cAMPS. (C) Glucagon secretion with increasing concentrations of cAMP. Glucagon and cAMP content were measured in islets exposed to increasing concentrations of forskolin (0-10 μM; concentrations (in μM) are given next to the data points) in the presence of 1 mM glucose. Data have been normalized to control (2.8±0.5 pg/islet/h; n=8). ***p<0.001 and **p<0.01 vs. secretion in the absence of forskolin. (D) Effects of increasing concentrations of adrenaline (5 pM-5 μM) on glucagon secretion. Grey rectangles indicate glucagon secretion at 1 mM glucose alone (top) and 8 mM glucose (bottom). Experiments were performed in the presence of 1 mM glucose. Data have been normalized to control (4.9±0.34 pg/islet/h; n=4). ***p<0.001 and *p<0.05 vs. secretion in the absence of adrenaline. (E) Confocal immunostaining of cells dispersed from single mouse islets. Cells were labelled with antibodies against glucagon (red) and PKA-RI (green). Rightmost panel shows the superimposed images (merge). (F) As in E but using antibody against PKA-RII. (G) Schematic illustration of image analysis. The ratio between near-plasma membrane and cytosolic immunoreactivity was determined using the equation inserted into the image. (H) Ratio between near-plasma membrane and cytosolic immunoreactivity calculated as illustrated in G. ***p<0.001 (n=10 for PKA-RI and PKA-RII.

Figure 3

Cyclic AMP-dependent modulation of the membrane potential dependence of glucagon secretion. (A) Membrane potential recordings from α-cells within intact islets (spontaneously active at 1 mM glucose) at 3.6 mM, 15 mM, 30 mM and 70 mM extracellular K⁺ (as indicated). (B) Glucagon secretion measured at extracellular K⁺ concentrations ([K⁺]_o) between 2.5 and 65 mM under control conditions (□) and in the presence of 10 μM forskolin (●). Glucose was present at 1 mM. The membrane potentials indicated (top) were obtained from experiments of the type as shown in A (n=7, 4, 4, 3 at 3.6 mM, 15 mM, 30 mM and 70 mM). Secretion data have been normalized to control (34.4±4.5 pg/islet/h measured at 4.7 mM [K⁺]_o; n=4-8). All values in the presence of forskolin are significantly different from corresponding control values (p<0.01 or better). Glucagon release under control conditions at 15 mM [K⁺]_o is significantly (p<0.05) lower, while glucagon secretion at 32 and 65 mM [K⁺]_o is significantly (p<0.001) higher than that at 4.7 mM [K⁺]_o. (C) Glucagon secretion measured at 4.7 mM, 15 mM and 65 mM [K⁺]_o (control, □) and in the presence of 5 μM adrenaline (●) or 100 nM GLP-1 (▲). Glucose was present at 1 mM. Data have been normalized to glucagon secretion at 4.7 mM [K⁺]_o (33.4±1.5 pg/islet/h; n=10). ***p<0.001 for adrenaline vs. control and †††p<0.001 for GLP-1 vs. control. (D) Changes in membrane capacitance (ΔC_m) displayed against membrane potential of depolarization (V) under control conditions (□) and 4 min after application of 10 μM forskolin (●). n=5 cells. *p<0.05; **p<0.01 vs. control. The inset shows the response to a depolarization to −20 mV. (E) As in D but comparing responses in the presence of 5 μM adrenaline (●) with control responses (□). n=5 cells. *p<0.05; **p<0.01; ***p<0.001 vs. control.

Figure 4

Involvement of Epac2 in α-cell exocytosis and glucagon secretion. (A) Changes in membrane capacitance (ΔC_m) elicited by voltage-clamp depolarization from −70 mV to −10 mV under control conditions (Ctrl), in the presence of 0.1 mM of the Epac2 agonist 8CPT-2Me-cAMP (8-CPT) and in the simultaneous presence of 8CPT-2Me-cAMP and 2 μM isradipine (Isr + 8-CPT). (B) Changes in membrane capacitance (ΔC_m) displayed against membrane potential of depolarization (V) under control conditions (■), after inclusion of 0.1 mM 8CPT-2Me-cAMP in the intracellular medium (○), and in 8CPT-2Me-cAMP containing cells exposed to 2 μM isradipine (). Data are mean values ± S.E.M. of 7-13 experiments. *p<0.05, **p<0.01 and ***p<0.001 for comparisons between 8CPT-2Me-cAMP alone or 8CPT-2Me-cAMP in the simultaneous presence of isradipine vs. control. ††p<0.01 for values in simultaneous presence of 8CPT-2Me-cAMP and isradipine vs. 8CPT-2Me-cAMP alone. (C) Whole-cell Ca²⁺-currents recorded under control conditions (Ctrl), after intracellular application of 100 μM 8-CPT-2Me-cAMP (8-CPT) and in the presence of 8-CPT and 2 μM isradipine. (D) Peak Ca²⁺-currents recorded under control conditions (■), after intracellular addition of 8-CPT (○) and after intracellular application of 8-CPT when L-type Ca²⁺-channels were blocked by isradipine (2 μM). *p<0.05 and **p<0.01 for the stimulatory effects of 8-CPT (vs. Ctrl) and †p<0.05 and ††p<0.01 for the effect of isradipine (vs. 8-CPT). (n=5-10 experiments in each group) (E) Glucagon secretion from wildtype mouse islets under control conditions (Ctrl; 1 mM glucose), in the presence of 100 nM GLP-1 or 5 μM adrenaline (Adr) in the absence and presence of 10 μM 8-Br-Rp-cAMPS. n=6-8. **p<0.01 and ***p<0.001 vs. Ctrl in the absence or presence of 8-Br-Rp-cAMPS; ††p<0.01 and †††p<0.001 vs. corresponding value in the absence of 8-Br-Rp-cAMPS. Glucose was present at 1 mM. (F) As in C using islets from Epac2 null mice. n=5-8. **p<0.01 and ***p<0.001 vs. Ctrl in the absence or presence of 8-Br-Rp-cAMPS. ††p<0.05 vs. corresponding value in the absence of 8-Br-Rp-cAMPS.

Figure 5

Effects of GLP-1, adrenaline and Ca²⁺-channel blockers on α-cell [Ca²⁺]_i and glucagon secretion (A) Spontaneous [Ca²⁺]_i-oscillations in individual α-cells in intact mouse islets exposed to 1 mM glucose and effects of including 10 nM GLP-1 or 5 μM adrenaline in the perfusion medium during the periods indicated by horizontal lines. n=12 cells in 4 islets from 3 mice. (B) As in A but 100 nM ω-conotoxin and 2 μM isradipine were applied. n= 14 cells in 4 islets from 2 mice. (C) Glucagon secretion at 1 mM glucose (Ctrl) and in the presence of 10 μM forskolin in the absence (left) and presence of 1 μM ω-conotoxin (middle) or 50 μM nifedipine (right). Data have been normalized to control in the absence of forskolin and the Ca²⁺-channel blockers (30.7±1.2 pg/islet/h; n=8-10). ***p<0.001 vs. respective control (Ctrl) in the absence or presence of ω-conotoxin or isradipine. †††p<0.001 vs. corresponding value in the absence of ω-conotoxin or isradipine. (D) As in C but comparing the effects of 100 nM GLP-1, 100 nM GIP and 5 μM adrenaline (Adr) in the absence (left) and presence (right) of 100 nM ω-conotoxin. n=10. *p<0.05, **p<0.01 and ***p<0.001 vs. respective Ctrl in the absence or presence of ω-conotoxin; ††p<0.01 vs. corresponding value in the absence of ω-conotoxin. (E) As in D but 2 μM isradipine was applied. n=7-10. *p<0.05 and ***p<0.001 vs. Ctrl; †p<0.001 vs. GIP in absence of isradipine and †††p<0.001 vs. adrenaline in absence of isradipine.

Figure 6

Effects of GLP-1, ω-conotoxin, adrenaline and somatostatin on mouse α-cell electrical activity (A) Action potential firing in an α-cell in an intact mouse islet at 1 mM glucose before, during and after addition of 10 nM GLP-1 (horizontal line). (B) Examples of action potentials taken under control conditions (i), during the transient repolarization (ii) and at “steady-state” at the end of the GLP-1 application (iii). (C) As in A but testing the effects of 100 nM ω-conotoxin on an isolated α-cell. (D) Examples of action potentials recorded before (i) and after addition of ω-conotoxin (ii). (E) As in A but 5 μM adrenaline was applied. (F) Examples of action potentials under control conditions (i) and broad action potentials seen in the presence of adrenaline (ii) (G) As in A but testing the effects of 100 nM somatostatin. (H) Action potential recorded under control conditions (i) and when electrical activity had resumed in the continued presence of somatostatin (ii) In A,C, E and F, the dotted horizontal lines indicate zero mV (top) and −50 mV (lower).

Fig. 7

GLP-1 blocks N-type Ca²⁺-channels and inhibits exocytosis. (A) Whole-cell Ca²⁺-currents evoked by membrane depolarization from −70 mV to 0 mV under control conditions (Ctrl; 1 mM glucose), 5 min after addition of GLP-1 (10 nM) and 5 min after addition of 100 nM ω-conotoxin in the continued presence of GLP-1 (GLP-1 and ω-con; grey). (B) Current (I)-voltage (V) relationship recorded using the perforated patch whole-cell configuration under control conditions (□; n=10), 5 min after addition of 10 nM GLP-1 (●; n=10), and 5 min after addition of ω-conotoxin (100 nM: ω-con) in the continued presence of GLP-1 (; n=6). *p<0.05 for effects of GLP-1 vs. control. (C-D) As in A-B but recorded under control conditions (Ctrl, black), 6 min after the addition of 10 μM 8-Br-Rp-cAMPS (Rp; dark grey) and 4 min after addition of 100 nM GLP-1 in the continued presence of 8-Br-Rp-cAMPS (Rp and GLP-1; light gray) in α-cells (E) Changes in membrane capacitance (ΔC_m) elicited by ten voltage-clamp depolarizations from −70 mV to 0 mV under control conditions (1 mM glucose; □), after the addition of 10 μM 8-Br-Rp-cAMPS and in the simultaneous presence of 10 nM GLP-1 8-Br-Rp-cAMPS (●). (F) Histogram of the mean increase in membrane capacitance elicited by the entire train (Total) and increase evoked by the two first depolarizations (RRP). n=6; *p<0.05, **p<0.01.