Glucose-induced modulation of [cAMP]pm in alpha cells parallels changes in glucagon secretion
TIRF imaging of mouse islets expressing a cAMP biosensor and exposed to 1–3 mmol/l glucose showed that [cAMP]pm was stable in most cells. An increase in the glucose concentration to 20 mmol/l resulted in decrease of [cAMP]pm in cells identified as alpha cells by their positive [cAMP]pm response to 10 μmol/l adrenaline (Fig. 1a–c). The [cAMP]pm lowering started immediately or after a delay of up to 2.3 min. Half-maximal decrease was observed 1.9 ± 0.2 min after the start of the decline. The beta cells within the same islet usually responded with a [cAMP]pm increase after glucose elevation and often, but not always, with adrenaline-induced lowering (Fig. 1b, c). The effect of 7 mmol/l glucose on alpha cell [cAMP]pm was close to maximal and was often characterised by an initial nadir followed by a somewhat less pronounced sustained reduction (see ESM Results and ESM Fig. 2). Some cells showed additional decrease at 20 mmol/l glucose, but the mean effect did not reach statistical significance (Fig. 1d, e). Alpha cells within human islets showed similar [cAMP]pm reductions in response to glucose elevation (Fig. 1f–h). When the glucose concentration was instead lowered from 7 to 1 mmol/l, mouse alpha cells responded with a rise in [cAMP]pm (Fig. 1i) and perifusion experiments revealed stimulated glucagon secretion with strikingly similar kinetics (Fig. 1j). Control experiments in cAMP biosensor-expressing islet alpha cells loaded with the pH indicator BCECF ascertained that the cAMP responses to glucose did not reflect a pH effect on the biosensor (see ESM Results and ESM Fig. 3).
Glucose-induced changes in alpha cell [cAMP]pm show little correlation with [Ca2+]pm
As Ca2+ might influence cAMP by regulating adenylyl cyclases and phosphodiesterases, we investigated whether the changes in [cAMP]pm were secondary to those in [Ca2+]pm by simultaneously recording the messengers in the same cell. In the presence of 1–3 mmol/l glucose, alpha cells in intact islets typically exhibited fast, irregular [Ca2+]pm spiking (Fig. 2a, b). An increase in the glucose concentration to 7 and 20 mmol/l sometimes resulted in a reduced amplitude and frequency of the [Ca2+]pm spikes (Fig. 2b) but often lacked a clear effect, or [Ca2+]pm even increased, also when [cAMP]pm decreased in the same cell (Fig. 2a). Similarly, when the islets were exposed to a reduction in glucose from 7 to 1 mmol/l, [cAMP]pm increased without a clear effect on [Ca2+]pm (Fig. 2c). A link between the two messengers was nevertheless observed in occasional alpha cells. Fig. 2d exemplifies an alpha cell exposed to 7 mmol/l glucose in which slow [Ca2+]pm oscillations are accompanied by similar changes in [cAMP]pm, and Fig. 2e shows that the alpha-cell-characteristic [Ca2+]pm rise in response to glutamate at 1 mmol/l glucose  was sometimes associated with an increase in [cAMP]pm. In beta cells, [Ca2+]pm was low and stable at 1 mmol/l glucose. Elevation to 7 and 20 mmol/l glucose induced an initial lowering in [Ca2+]pm followed by concomitant increases in [Ca2+]pm and [cAMP]pm (Fig. 2f), consistent with previous observations .
[cAMP]pm lowering by glucose elevation occurs independently of paracrine insulin and somatostatin signalling
To test if [cAMP]pm is influenced by insulin or somatostatin, the hormones or antagonists of their receptors were added to the islets. Insulin (100 nmol/l) had little effect on alpha cell [cAMP]pm at 1 mmol/l glucose, whereas an increase in glucose to 20 mmol/l significantly reduced [cAMP]pm in the same cell (Fig. 3a). Inhibition of the action of endogenous insulin at 20 mmol/l glucose with the insulin receptor antagonist S961 (1 μmol/l) was also without effect (Fig. 3b), indicating that paracrine insulin signalling unlikely contributes to the [cAMP]pm reduction.
Somatostatin was more efficient and lowered [cAMP]pm at 100 pmol/l in three out of 12 cells from four experiments. Of the remaining alpha cells, seven responded to 1 nmol/l, whereas two cells showed [cAMP]pm reductions only after an increase in somatostatin to 100 nmol/l (data not shown). We therefore used 1 nmol/l somatostatin in the remaining experiments and found that 13 out of 16 alpha cells from six experiments responded at this concentration with a reduction in [cAMP]pm (Fig. 3c). The SSTR2 antagonist PRL2903 (5 μmol/l) often increased [cAMP]pm irrespective of the glucose concentration (7/11 alpha cells in three experiments at 1 mmol/l glucose and 7/9 cells in two experiments at 20 mmol/l glucose), but prevented neither [cAMP]pm lowering by increasing glucose from 1 to 7 and 20 mmol/l (Fig. 3d, e), nor inhibition of glucagon secretion by 5 and 20 mmol/l glucose (Fig. 3f). The experiment in Fig. 3f also shows that glucagon release was suppressed by 5 mmol/l glucose without any simultaneous stimulation of insulin secretion, reinforcing that paracrine signalling from beta cells is not important under those conditions.
Another SSTR2 antagonist, CYN 154806, also increased [cAMP]pm at 1 mmol/l glucose in many alpha cells, but did not prevent [cAMP]pm reduction induced by a subsequent elevation in the glucose concentration (Fig. 3g). To exclude the involvement of somatostatin receptors other than SSTR2, we treated the islets with pertussis toxin (200 ng/ml for 18 h) to inhibit the action of Gαi, which transduces signals from all somatostatin receptor subtypes. Pertussis toxin did not affect the [cAMP]pm increase induced by a reduction in the glucose concentration from 7 to 1 mmol/l, but prevented the [cAMP]pm-lowering effect of somatostatin (Fig. 3h–j).
Glucose-induced cAMP modulation involves an alpha-cell-intrinsic mechanism
To reinforce the conclusion that glucose modulates cAMP in alpha cells, we recorded cAMP with an alternative sensor, the FRET-based Epac-SH188 . Confocal detection of the FRET ratio showed that an increase in glucose from 1 to 7 mmol/l triggered lowering of [cAMP] throughout the cytoplasm of islet alpha cells (Fig. 4a). FRET detection with TIRF imaging indicated glucose-induced [cAMP]pm changes similar to those recorded with the translocation sensor, and repeated reductions of glucose from 7 to 1 mmol/l induced recurring [cAMP]pm elevations (Fig. 4b). To consolidate the conclusion that the glucose effect was independent of auto- and paracrine signalling, Ca2+ was omitted and 2 mmol/l EGTA added to prevent secretion from all endocrine islet cells. There was a slight increase in [cAMP]pm upon Ca2+ removal in the presence of 7 mmol/l glucose (Fig. 4c), possibly reflecting disappearance of paracrine inhibitory signals. Glucose reduction to 1 mmol/l still induced a pronounced [cAMP]pm elevation, although the amplitude was slightly lower than that in the presence of Ca2+ (Fig. 4d).
In an additional approach to prevent paracrine signalling, experiments were performed after dispersion of islets into single cells. Individual alpha cells responded to a 7 to 1 mmol/l reduction in glucose or the addition of adrenaline with increases in [cAMP]pm similar to those in islets (Fig. 4e, f). In addition, dispersed beta cells showed islet-similar responses to glucose reduction and adrenaline, with lowering of [cAMP]pm (Fig. 4g).
Glucose-induced inhibition of glucagon secretion occurs independently of somatostatin and is prevented by fixing cAMP at high levels
Next, we investigated how cAMP-modulating agents influence glucagon secretion. In batch-incubated islets, the elevation of glucose from 1 to 7 or 20 mmol/l inhibited glucagon secretion by more than 80%, but in the presence of the membrane-permeable cAMP analogue 8-Br-cAMP secretion remained high independent of the glucose concentration (Fig. 5a). To avoid the variability among groups inherent to batch incubations, we employed an alternative approach based on islet perifusion, allowing sequential exposure of the same islets to different test conditions. This perifusion approach with low temporal resolution was first used to compare the glucagon responses to two successive challenges with 7, 1 and 20 mmol/l glucose on the day of islet isolation (day 0) with islets from the same mouse that had been cultured until the next day (day 1). After 1 day of culture, the glucagon responses to the 1 to 7 mmol/l glucose transitions were more pronounced and reproducible, with approximately eightfold increases compared with four- to fivefold increases on day 0 (Fig. 5b, c). It is possible that higher unstimulated and stimulated secretion on day 0 reflect dysregulated glucagon release after cell perturbation during islet isolation.
Using the perifusion approach with overnight-cultured islets, we investigated how somatostatin influences glucose- and cAMP-regulated glucagon secretion. SSTR2 inhibition with CYN 154806 increased glucagon release at 7 mmol/l glucose but did not prevent 1 mmol/l glucose from inducing a similar increase in secretion as in the absence of the drug (Fig. 5d). After pertussis toxin treatment, the islets showed higher initial glucagon secretion at 7 mmol/l glucose and CYN 154806 lacked a significant effect, but the secretory responses to glucose changes were not different from those of the control (Fig. 5e). Fixing cAMP high with 100 μmol/l of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) in the presence of CYN 154806 to evade the influence of somatostatin prevented inhibition of glucagon release in response to a 1 to 7 mmol/l glucose transition (Fig. 6a), underscoring that glucose acts by decreasing cAMP.
PKA inhibition mimics glucose inhibition of glucagon secretion
Since many effects of cAMP are mediated by PKA, we investigated the potential involvement of this kinase in glucagon secretion. Changing glucose from 7 to 1 mmol/l induced a marked stimulation of glucagon secretion (Fig. 6b). Subsequent introduction of the PKA inhibitor Rp-8-CPT-cAMPS (100 μmol/l) reduced secretion by more than 70%, and there was some further reduction at 7 mmol/l of the sugar (Fig. 6b).