Effects of adenosine A(1) receptor antagonism on insulin secretion from rat pancreatic islets.

Abstract

Adenosine is known to influence different kinds of cells, including beta-cells of the pancreas. However, the role of this nucleoside in the regulation of insulin secretion is not fully elucidated. In the present study, the effects of adenosine A(1) receptor antagonism on insulin secretion from isolated rat pancreatic islets were tested using DPCPX, a selective adenosine A(1) receptor antagonist. It was demonstrated that pancreatic islets stimulated with 6.7 and 16.7 mM glucose and exposed to DPCPX released significantly more insulin compared with islets incubated with glucose alone. The insulin-secretory response to glucose and low forskolin appeared to be substantially potentiated by DPCPX, but DPCPX was ineffective in the presence of glucose and high forskolin. Moreover, DPCPX failed to change insulin secretion stimulated by the combination of glucose and dibutyryl-cAMP, a non-hydrolysable cAMP analogue. Studies on pancreatic islets also revealed that the potentiating effect of DPCPX on glucose-induced insulin secretion was attenuated by H-89, a selective inhibitor of protein kinase A. It was also demonstrated that formazan formation, reflecting metabolic activity of cells, was enhanced in islets exposed to DPCPX. Moreover, DPCPX was found to increase islet cAMP content, whereas ATP was not significantly changed. These results indicate that adenosine A(1) receptor blockade in rat pancreatic islets potentiates insulin secretion induced by both physiological and supraphysiological glucose concentrations. This effect is proposed to be due to increased metabolic activity of cells and increased cAMP content.

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Effects of adenosine A1 receptor antagonism on insulin secretion
from rat pancreatic islets


Agnieszka Zywert, Katarzyna Szkudelska and Tomasz Szkudelski*





Short title: Adenosine A1 receptor antagonism and insulin secretion


*- corresponding author
Department of Animal Physiology and Biochemistry
Poznan University of Life Sciences
Wolynska 35, 60-637 Poznan, Poland
tel/fax: +48 61 8487197
e-mail: tszkudel@jay.up.poznan.pl

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Summary
Adenosine is known to influence different kinds of cells, including -cells of the pancreas.
However, the role of this nucleoside in the regulation of insulin secretion is not fully
elucidated. In the present study, the effects of adenosine A1 receptor antagonism on insulin
secretion from isolated rat pancreatic islets were tested using DPCPX, a selective adenosine
A1 receptor antagonist. It was demonstrated that pancreatic islets stimulated with 6.7 and 16.7
mM glucose and exposed to DPCPX released significantly more insulin compared with islets
incubated with glucose alone. The insulin-secretory response to glucose and low forskolin
appeared to be substantially potentiated by DPCPX, however, in the presence of glucose and
high forskolin, DPCPX was ineffective. Moreover, DPCPX failed to change insulin secretion
stimulated by the combination of glucose and dibutyryl-cAMP, a non-hydrolysable cAMP
analogue. Studies on pancreatic islets also revealed that the potentiatory effect of DPCPX on
glucose-induced insulin secretion was attenuated by H-89, a selective inhibitor of protein
kinase A.
It was also demonstrated that formazan formation, reflecting metabolic activity of cells, was
enhanced in islets exposed to DPCPX. Moreover, DPCPX was found to increase islet cAMP
content, whereas ATP was not significantly changed.
These results indicate that adenosine A1 receptor blockade in rat pancreatic islets potentiates
insulin secretion induced by both physiological and supraphysiological glucose. This effect is
proposed to be due to increased metabolic activity of cells and increased cAMP content.


Key words: insulin, secretion, adenosine, islets

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Introduction
Under physiological conditions, insulin secretion is precisely regulated according to the actual
demand of the organism. Glucose is the main physiological stimulator of insulin secretion.
The insulinotropic action of glucose is preceded by a sequence of events involving its
transport by facilitated diffusion and its oxidative metabolism, an increase in ATP/ADP ratio,
closure of ATP-dependent potassium channels, membrane depolarisation, opening of voltage-
dependent calcium channels and increase in cytosolic calcium. Finally, the rise in cytosolic
calcium concentration triggers secretion of insulin. Moreover, additional signals in -cells are
generated to maintain the sustained secretory response to glucose (Henquin 2000, 2009).
The insulin-secretory response to glucose is known to be influenced by different stimulatory
and inhibitory factors, such as dietary compounds (Newsholme et al. 2005, Nolan et al. 2006,
Pinent et al. 2008), nervous system (Ahrén 1990) and incretins (Ahrén 2003, Baggio and
Drucker 2007). Moreover, secretion of insulin undergoes paracrine regulation by glucagon
and somatostatin (Schatz and Kullek 1980).
It is also known that the endocrine pancreas is influenced by purines (Petit et al. 2009).
Effects induced by these compounds are mediated via purinergic receptors. Different types of
purinergic receptors had been described in the pancreatic islet cells. Among them, adenosine
A1 receptor was identified indicating the potential regulatory role of adenosine in insulin
release (Hillaire-Buys et al. 1987, 1994). Experimental data confirmed this assumption. It is
known that in β-cells ATP is hydrolyzed to adenosine and this nucleoside is released from the
cell, binds adenosine A1 receptor, which is negatively coupled to adenylate cyclase, and
insulin secretion is inhibited (Bertrand et al. 1989a, Hillaire-Buys et al. 1989). However, the
physiological relevance of islet-derived adenosine in the regulation of β-cells is not fully
elucidated. Studies on mice demonstrated that the magnitude of the second phase of insulin
secretion was significantly greater in the case of pancreata obtained from adenosine A1

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receptor deficient animals compared with secretion observed in normal mice (Johansson et al.
2007). On the other hand, experiments on isolated pancreatic islets revealed that adenosine
may exert an opposite effect on insulin secretion depending on nucleoside concentration. At
high concentrations, adenosine was found to enhance insulin release, whereas at lower
concentrations an inhibition was reported. The inhibition of hormone secretion is thought to
result from the action of adenosine via adenosine A1 receptor, whereas the opposite effect is
proposed to be due to the intracellular metabolism of the nucleoside (Bertrand et al. 1989a,
Petit et al. 2009). Data from the literature on the role of adenosine in the regulation of insulin
secretion are not fully coherent since different effects were observed depending on nucleoside
concentration, animal species and other experimental conditions (Ismail et al. 1977, Campbell
and Taylor 1982, Bertrand et al. 1989a, Bertrand et al. 1989b, Petit et al. 1989, Verspohl et al.
2002, Töpfer et al. 2008, Hillaire-Buys et al. 1989). The aim of the present study was to
determine the effects of adenosine A1 receptor antagonism on insulin secretion from rat
pancreatic islets.


Methods

Animals
Male Wistar rats weighing 280-350 g obtained from Brwinow (Poland) were used in all
experiments. The rats were fed ad libitum a standard laboratory diet (Labofeed, Kcynia,
Poland) and had free access to tap water. The animals were maintained in cages in an air-
conditioned room with a 12:12-h dark-light cycle and a constant temperature 21 ±1 °C. The
rats were killed by decapitation. The experiments were performed according to rules accepted
by Local Ethical Commission for Investigation on Animals.

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Islet isolation
Pancreatic islets were isolated by a collagenase digestion according to Lacy and
Kostianovsky (1967). Hanks’ solution (containing in mM: NaCl 137, KCl 5.36, MgSO4 0.81,
Na2HPO4 0.34, KH2PO4 0.44, CaCl2 1.26, NaHCO3 4.17) gassed with 95% O2/5% CO2 was
used during the isolation. The solution was injected into the common bile duct and the
pancreas was excised. In each experiment, glands obtained from three rats were pooled, cut
down with scissors and incubated with collagenase. Afterwards, islets were washed with
Hanks’ solution without the enzyme and were separated from the remaining exocrine tissue
by hand picking under a stereomicroscope.

The effects of adenosine A1 receptor blockade on insulin secretion
To study the effects of adenosine A1 receptor blockade on insulin secretion, groups of 5 islets
were incubated in 1 ml of Krebs-Ringer buffer (containing in mM: 115 NaCl, 24 NaHCO3, 5
KCl, 1 MgCl2, 1 CaCl2 and 0.5% bovine serum albumin). Before use the buffer was gassed
with 95% O2/5% CO2 and its pH was adjusted to 7.4. In each experiment, islets were
incubated for 90 min in a water bath at 37 °C in an atmosphere of O2/CO2 (95%/5%) with a
gentle shaking. In the experiments, pancreatic islets were incubated in the presence of 2.8
mM glucose (basal secretion of insulin), 6.7 mM glucose (physiological concentration) or
16.7 mM glucose (supraphysiological concentration).
In the first part of our experiments, the effects of adenosine A1 receptor blockade on insulin
secretion induced by physiological and supraphysiological glucose were studied. For this
purpose, pancreatic islets were stimulated with 6.7 or 16.7 mM glucose alone or glucose in
the presence of 1, 10 and 50 M DPCPX.