Elsevier

Cellular Signalling

Volume 61, September 2019, Pages 48-56
Cellular Signalling

PACAP stimulates insulin secretion by PAC1 receptor and ion channels in β-cells

https://doi.org/10.1016/j.cellsig.2019.05.006Get rights and content

Highlights

  • PACAP38 amplifies glucose-induced insulin secretion in rat pancreatic islets.

  • PACAP38 stimulates insulin secretion by PAC1 receptor and AC/PKA signaling pathway.

  • PACAP38 inhibits Kv channels by regulating PAC1-R and AC/PKA signaling.

  • PACAP38 activates voltage-dependent Ca2+ channels via PAC1-R.

  • PACAP38 increases intracellular Ca2+ concentration through PAC1-R.

Abstract

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays a crucial role in the endocrine system. The present study aimed to investigate the effect of PACAP38 on insulin secretion and the underlying mechanism in rat pancreatic β-cells. The insulin secretion results showed that PACAP38 stimulated insulin secretion in a glucose- and dose-dependent manner. The insulinotropic effect was mediated by PAC1 receptor, but not by VPAC1 and VPAC2 receptors. Inhibition of adenylyl cyclase and protein kinase A suppressed PACAP38-augmented insulin secretion. Glucose-regulated insulin secretion is dependent on a series of electrophysiological activities. Current-clamp technology suggested that PACAP38 prolonged action potential duration. Voltage-clamp recordings revealed that PACAP38 blocked voltage-dependent potassium currents, and this effect was reversed by inhibition of PAC1 receptor, adenylyl cyclase, or protein kinase A. Activation of Ca2+ channels by PACAP38 was also observed, which could be antagonized by the PAC1 receptor antagonist. In addition, calcium-imaging analysis indicated that PACAP38 increased intracellular Ca2+ concentration, which was decreased by PAC1 receptor antagonist. These findings demonstrate that PACAP38 stimulates glucose-induced insulin secretion mainly by acting on PAC1 receptor, inhibiting voltage-dependent potassium channels, activating Ca2+ channels and increasing intracellular Ca2+ concentration. Further, PACAP blocks voltage-dependent potassium currents via the adenylyl cyclase/protein kinase A signaling pathway.

Introduction

Pituitary adenylate cyclase-activating polypeptide (PACAP), a hypothalamic peptide, activates adenylate cyclase (AC) in rat pituitary cells [1]. PACAP is a member of the secretin-glucagon superfamily [2], including a 38-amino-acid form (PACAP38) and a shorter 27-amino-acid form (PACAP27). Chromatographic analyses have shown that the concentration of PACAP38 in extracted human pancreas is much higher than that of PACAP27 [3,4]. The peptide elicits its biological actions via regulating three types of class II G protein-coupled receptors (GPCRs): PAC1 receptor (PAC1-R), VPAC1 receptor (VPAC1-R), and VPAC2 receptor (VPAC2-R) [5]. PAC1-R has a distinct high affinity for PACAP. Study has shown that PACAP and its receptors are widely distributed in the central nervous system (CNS) and peripheral organs [1].

Previous study suggested that PACAP is a potential therapeutic target for diabetes and its complications [5]. It has been proposed that PACAP regulates postprandial glucose homeostasis and potentiates glucose-dependent insulin secretion from isolated rat islets [6,7]. However, the underlying mechanism of this action is still unknown. Pancreatic β-cells possess electrical excitability. Glucose-induced insulin secretion from β-cells is mediated by a series of electrophysiological activities, which result in exocytosis of insulin-containing granules [8]. Hence, we proposed that electrogenic events were involved in PACAP38-modulated glucose-stimulated insulin secretion through acting on G protein-coupled receptor and AC/PKA signaling pathways.

In this study, we used primary rat pancreatic islets and dispersed islet cells to explore the effects of PACAP38 on β-cell function, including insulin secretion, electrical activity, and intracellular Ca2+ concentration. Based on insulin secretion experiments and patch-clamp technology, we identified the cellular and molecular mechanisms whereby PACAP38 amplified glucose-induced insulin secretion.

Section snippets

Animals

Adult male Wistar rats, weighing 240–260 g, were purchased from Beijing Weitong Lihua experimental animal center. Rats were housed with pellet-type food and tap water under temperature (25 ± 2 °C) and humidity (55–60%) condition with a 12 h-light/darkness cycle. All experimental procedures involving animals described below were in accordance with the ethical guidelines for animal research at Shanxi Medical University and were approved by the Animal Care and Use Committee of Shanxi Medical

PACAP38 amplifies glucose-induced insulin secretion in rat pancreatic islets

To examine the effect of PACAP38 on insulin secretion, islets were stimulated with different doses of PACAP38 under 2.8 or 8.3 mM glucose. As shown in Fig. 1A, PACAP38 at various concentrations (0–100 nM) had no effect on insulin secretion under 2.8 mM glucose conditions. However, under conditions of 8.3 mM glucose, PACAP38 (10 and 100 nM) potentiated insulin secretion from isolated rat islets.

To confirm whether PACAP38-induced insulin secretion was glucose-dependent, islets were stimulated

Discussion

PACAP is a widely known potential insulinotropic neuropeptide [21,22]. It has been reported that administration of PACAP in vivo significantly enhanced plasma insulin levels in mice [23], pigs [24], and humans [25]. And studies have reported that PACAP amplified insulin secretion in a glucose-dependent manner [21,26]. Of note, we found that PACAP38 had no effect on insulin secretion at a low glucose concentration. PACAP38 dose-dependently augmented insulin secretion under high glucose

Declaration of interest

None.

Author contribution statement

Y Z, Y L and M L conceived and designed the study; M L, X Y, T B, Z L, T L and L C carried out the experiments; M L, X Y and T B contributed to analyze the data and interpret the results of the experiments; Y Z, Y L and M L wrote the manuscript; Y Z, Y L, M L and Y W revised the manuscript. All authors read and approved the final version.

Acknowledgments

This work was supported by NSFC (81670710; 81770776), Advanced Programs of Shanxi for the Returned Overseas Chinese Scholars (2016-97), Research Project Supported by Shanxi Scholarship Council of China (2017-053),FSKSC and 1331KSC.

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