Effects of the β-carbolines, harmane and pinoline, on insulin secretion from isolated human islets of Langerhans

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Abstract

It is well known that certain imidazoline compounds can stimulate insulin secretion and this has been attributed to the activation of imidazoline I3 binding sites in the pancreatic β-cell. Recently, it has been proposed that β-carbolines may be endogenous ligands having activity at imidazoline sites and we have, therefore, studied the effects of β-carbolines on insulin secretion. The β-carbolines harmane, norharmane and pinoline increased insulin secretion two- to threefold from isolated human islets of Langerhans. The effects of harmane and pinoline were dose-dependent (EC50: 5 and 25 μM, respectively) and these agents also blocked the inhibitory effects of the potassium channel agonist, diazoxide, on glucose-induced insulin release. Stimulation of insulin secretion by harmane was glucose-dependent but, unlike the imidazoline I3 receptor agonist efaroxan, it increased the rate of insulin release beyond that elicited by 20 mM glucose (20 mM glucose alone: 253±34% vs. basal; 20 mM glucose plus 100 μM harmane: 327±15%; P<0.01). Stimulation of insulin secretion by harmane was attenuated by the imidazoline I3 receptor antagonist KU14R (2 (2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole) and was reduced when islets were treated with efaroxan for 18 h, prior to the addition of harmane. The results reveal that β-carbolines can potentiate the rate of insulin secretion from human islets and suggest that these agents may be useful prototypes for the development of novel insulin secretagogues.

Introduction

It is now well established that the pancreatic β-cell is equipped with a class of imidazoline binding sites involved in the control of insulin secretion (reviewed by Efendic et al., 2002, Eglen et al., 1998, Morgan and Chan, 2001, Morgan et al., 1999, Rustenbeck et al., 1999). These sites appear to belong to the larger family of imidazoline binding proteins that are expressed both in the central nervous system and in peripheral tissues Bousquet, 2000, Bousquet, 2001, Bousquet et al., 2000, Eglen et al., 1998, Ernsberger et al., 1997, Head et al., 1998, Khan et al., 1999, Molderings and Gothert, 1999, Parini et al., 1996, Regunathan and Reis, 1996. However, the β-cell imidazoline binding sites exhibit a number of differences from those described elsewhere and, in recognition of this, they have been designated “I3” to differentiate them from the more ubiquitous imidazoline “I1” and “I2” sites Chan et al., 1994, Eglen et al., 1998, Morgan and Chan, 2001.

Binding of agonists to the imidazoline I3 site in β-cells leads to an increase in insulin secretion and compounds acting at this site have attracted attention as potential therapeutic agents for use in the management of type 2 diabetes Chan and Morgan, 1990, Chan et al., 2001, Efanov et al., 2001, Efanov et al., 2002, Efendic et al., 2002, Mest et al., 2001, Morgan and Chan, 2001. In addition, attention has also been drawn to the exciting possibility that an endogenous ligand may exist which interacts with the β-cell imidazoline I3 site and is involved in the physiological control of insulin secretion (Chan et al., 1997). Strong evidence for the existence of such a molecule has been provided from experiments in which extracts of rat brain were found to stimulate insulin secretion Chan, 1998, Chan et al., 1997. Clearly, identification of the active compound would have major implications for drug design.

Despite extensive work, the identity of the putative endogenous imidazoline binding site ligand has proved difficult to establish Atlas, 1995, Parker et al., 1999a, Parker et al., 1999b, Parker et al., 2000, Reis et al., 1995, Singh et al., 1995. The active principle has become known as “clonidine displacing substance” because of its capacity to compete with [3H]-clonidine in radioligand binding assays (Atlas, 1995) and a considerable weight of evidence has accumulated to suggest that clonidine displacing substance is a genuine physiological entity. Indeed, multiple clonidine displacing substance-like molecules may exist and their presence has been inferred in various tissue samples and body fluids Dontenwill et al., 1993, Meeley et al., 1992, Piletz et al., 1995, Parker et al., 1999a, Parker et al., 1999b, Parker et al., 2000, Reis et al., 1995, Reis et al., 1998, Singh et al., 1995, Wang et al., 1997.

The first structure to be proposed for clonidine displacing substance came from Li et al. (1994) who suggested that the activity may reside in a decarboxylated derivative of arginine, agmatine. These authors described the presence of agmatine in extracts of mammalian brain Li et al., 1994, Li et al., 1995, Raasch et al., 1995 and it was subsequently confirmed that agmatine behaves as a clonidine displacing substance in some, though not all, assay systems Berdeu et al., 1996, Chan et al., 1995, Gao et al., 1995, Herman, 1997, Li et al., 1994, Li et al., 1995, Pinthong et al., 1995, Raasch et al., 2002. The same group also established that pathways for synthesis and degradation of agmatine are present in certain brain regions Li et al., 1994, Li et al., 1995 and has provided firm evidence that agmatine may be a physiologically relevant molecule. However, it has also become clear that, although agmatine is present in pancreatic islet cells (Stickle et al., 1996), it does not fulfil the criteria expected of a ligand active at the imidazoline binding sites involved in control of insulin secretion Chan et al., 1995, Berdeu et al., 1996, Sener et al., 1989. Thus, an additional compound(s) must account for the insulin-secreting activity found in crude clonidine displacing substance preparations.

We have been conducting studies aimed to characterise the active components of crude clonidine displacing substance preparations more fully and have developed a fractionation method which provides improved resolution of the constituents Parker et al., 1999a, Parker et al., 1999b, Parker et al., 2000. Using radioligand binding studies to identify potentially active compounds, it was suggested that at least part of the clonidine displacing substance activity may be due to the presence of one or more agents having a β-carboline structure Husbands et al., 2001, Ruiz-Durantez et al., 2001, Musgrave and Badoer, 2000. Until recently, β-carbolines have not been considered as potential endogenous imidazoline binding site ligands but work by Musgrave and Badoer (2000) has revealed that microinjection of one β-carboline, harmane, into the rostral ventrolateral medulla of the rat leads to a hypotensive response. This is consistent with the possibility that harmane (or a derivative) may exert “clonidine displacing substance-activity” in the brain and suggests that harmane may be active at imidazoline binding sites located in the rostral ventrolateral medulla.

Despite this evidence from rat studies, it is still not clear whether β-carbolines are active at imidazoline sites expressed in human tissues nor whether they can interact functionally with similar sites located outside the central nervous system. In order to clarify these issues, we have examined the effects of two β-carbolines, harmane and pinoline, on insulin secretion from isolated rat and human islets of Langerhans. We have investigated whether these agents can stimulate insulin secretion and have attempted to establish whether the secretory response could reflect their interaction with a β-cell imidazoline binding site.

Section snippets

Materials

Collagenase (type XI), flurbiprofen, indomethacin, diazoxide, dimethylsulphoxide (DMSO), harmane, pinoline and norharmane were purchased from Sigma (Dorset, UK). Efaroxan hydrochloride was generously provided by RBI (Natick, MA, USA). KU14R (2 (2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole) was synthesised in the Dept. of Chemistry, University of Keele, UK (Clews et al., 2001) and is available from Tocris (Bristol, UK). [125I]-insulin was from Biogenesis and anti-bovine insulin antiserum (for

Effects of harmane on insulin secretion in the absence or presence of diazoxide

Initially, we examined whether β-carbolines have the ability to stimulate insulin secretion from isolated human islets of Langerhans and observed (Fig. 1) that three such molecules, harmane, norharmane and pinoline, all caused a significant, two- to threefold, increase in insulin secretion from islets incubated with 6 mM glucose. The magnitude of this increase was similar to that elicited by efaroxan, a well-characterised ligand for the β-cell imidazoline I3 site (Fig. 1). It is known that

Discussion

The recent demonstration that a β-carboline, harmane, can elicit hypotension when injected into rat brain (Musgrave and Badoer, 2000) has fuelled speculation that this, or a closely related molecule may, despite being structurally unrelated to imidazolines, represent one of the long-sought endogenous ligands for the class of “imidazoline” binding sites. In the present work, we have examined this proposition in the pancreatic β-cell, which expresses a pharmacologically atypical imidazoline site,

Acknowledgments

We gratefully acknowledge the financial support of Roche Bioscience, The Wellcome Trust and Diabetes UK.

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