Introduction
The pancreatic islets provide a centre where signals indicating the nutritional status of the body, including factors such as enteroendocrine hormones, nutrients, metabolites and neuronal signals, can converge and initiate changes in pancreatic hormone secretion to regulate blood glucose levels. Insulin (released from beta cells) and glucagon (released from alpha cells) exert opposite effects on glycaemia, with insulin promoting glucose uptake in conditions of high glucose and glucagon initiating hepatic glucose production in periods of decreasing glucose levels [
1]. Nuanced interactions and crosstalk between islet cell types are critical in maintaining tight control over blood glucose equilibrium, and elucidating the ways in which enteric signals and islet cells interact to influence circulating glucose levels could provide insights into the mechanisms underlying altered glycaemic control and diabetes [
2,
3].
A key paracrine mediator within islet cells is somatostatin (SST), which is produced by pancreatic delta cells. SST appears to exert tonic suppression of insulin and glucagon release within islets [
4]. The importance of this potent paracrine mechanism is illustrated by experiments showing that whole-animal genetic ablation of
Sst results in aberrant secretion of both insulin and glucagon from isolated islets in response to glucose [
5]. Indeed, the dysregulation of SST-mediated negative-feedback loops has been implicated in the development of type 2 diabetes [
6]. Compared with our knowledge of insulin and glucagon release, there is still much to learn about the regulatory pathways and cellular machinery underlying SST secretion. Identifying how delta cells differ from their neighbouring alpha and beta cells is crucial for interpreting transcriptomic and functional data obtained from whole islets [
7].
Ghrelin is a peptide hormone that has been identified as a key component of the gut–brain axis [
8]. It is synthesised predominantly in the stomach [
9,
10] and gastrointestinal tract [
11], although there have been reports of ghrelin-producing epsilon cells in adult islets [
12,
13]. Ghrelin levels in plasma are influenced by nutritional status and may influence growth hormone secretion, appetite and fat deposition [
14]. Importantly, there are indications that ghrelin plays a role in the regulation of the pancreas in response to changes in glucose levels [
15]. A large number of reports have examined the effects of the active acylated form of ghrelin on glucose-stimulated insulin secretion. The consensus of these studies is that ghrelin exerts acute inhibition of insulin release [
16‐
19], and that ghrelin infusions lead to impaired glucose tolerance [
20,
21]. In addition, pharmacological inhibition of ghrelin acylation (which is essential for the biological activity of ghrelin) via blockade of ghrelin
O-acyltransferase results in significant increases in glucose-stimulated insulin secretion and improves overall glucose tolerance [
22].
The cognate receptor for ghrelin is the growth hormone secretagogue receptor (GHSR) [
9]. The effects of ghrelin on insulin release are purportedly through direct receptor-mediated modulation of beta cell activity [
23,
24]. However, the predominant G
αq coupling of GHSR [
25] and the insulinostatic effects of ghrelin, if indeed mediated directly via beta cells, are paradoxical. Because of the therapeutic potential of manipulating the ghrelin axis in individuals with obesity and diabetes [
26], the mechanism by which ghrelin inhibits insulin release warrants further exploration.
The aims of this study were to build a transcriptomic profile of pancreatic delta cells, in comparison with alpha and beta cells, and to identify specific delta cell markers and regulators. Having demonstrated Ghsr expression to be highly enriched in delta cells, we further aimed to characterise the effects of ghrelin on delta cell signalling pathways and islet cell secretory profiles.
Discussion
In this study, we identified the transcriptome of pancreatic delta cells and performed a comparative transcriptomic analysis with beta and alpha cells. Amongst islet-expressed Gpcrs, we found Ghsr to be significantly enriched in delta cells over both alpha and beta cells. Although we were unable to identify antibodies suitable for confirming GHSR localisation at the protein level, the functional relevance of this receptor in delta cells was confirmed by the finding that GHSR agonism elicited increases in cytosolic calcium levels in isolated delta cells, and that in the perfused pancreas, ghrelin stimulated SST release while attenuating insulin and glucagon release in an SSTR-sensitive manner.
A multitude of studies involving genetic and pharmacological manipulation of GHSR have concluded that the action of ghrelin on glucose tolerance and glycaemia is reliant on GHSR binding and that its blockade, even on a high-fat background, improves glucose handling [
12,
16,
36‐
38]. Previous studies have concluded that the effects of ghrelin on insulin secretion are mediated by its direct binding to GHSR located on the beta cell plasma membrane, but the underlying signalling mechanism is difficult to explain. GHSR is predominantly G
αq coupled, so, like other beta cell G
αq-coupled receptors such as the muscarinic receptor M3, its activation would be predicted to enhance rather than inhibit insulin secretion. However, GHSR blockade in isolated islets has been reported to increase insulin release and cytosolic calcium in beta cells via a pertussis toxin-sensitive pathway [
16], implicating G
i/o G proteins [
39], and to be impaired by antisense oligonucleotides against G
αi2 [
23]. Administration of pertussis toxin has also been reported to render ghrelin incapable of lowering plasma insulin levels in vivo [
23].
To account for the paradoxical G
i/o dependence of a response downstream of a G
αq-coupled receptor, some have suggested non-canonical coupling of the ghrelin receptor to G
i/o G proteins via recruitment and heteromerisation of GHSR with SSTR5 in beta cell lines [
24]. However, our transcriptomic analysis found negligible expression of
Ghsr and
Sstr5 in mouse beta cells. This is unlikely to reflect technical limitations, as we have previously been able to detect
Sstr2,
Sstr3 and
Sstr5 in intestinal L cells [
40]. In the context of pancreatic islets, our data confirm relatively specific expression of
Sstr2 in alpha cells, but the high expression of
Sstr3 was unexpected [
41], suggesting that conclusions based on SSTR-selective agents and antibodies should be revisited. A recent study reported that re-expression of
Ghsr specifically in beta cells on a
Ghsr
–/– background rescued the ability of a GHSR antagonist to enhance glucose-stimulated insulin release during a glucose tolerance test [
42], supporting the direct detection of ghrelin by beta cells and suggesting that even extremely low levels of
Ghsr expression might modulate beta cell activity. To explain the GHSR-mediated suppression of insulin release and the involvement of a G
i/o-dependent pathway, our findings alternatively suggest that the inhibitory effect of ghrelin on insulin release is not entirely mediated directly via the beta cell, but instead proceeds at least in part by the activation of GHSR on delta cells, triggering SST release that subsequently inhibits beta cells through SSTR activation. Similar conclusions were reached in a paper submitted while this manuscript was under review [
43].
Our work presents an exhaustive transcriptomic comparison between murine pancreatic alpha, beta and delta cells (available at
www.ncbi.nlm.nih.gov/geo), providing a database for identifying factors that similarly or uniquely regulate different islet cell types. The transgenes used to fluorescently label alpha and delta cells did not alter islet architecture or the relative proportions of islet cell types [
30], but we cannot rule out the possibility that they had subtle effects on gene expression. Several recent studies have similarly analysed the gene-expression profiles of pancreatic alpha and beta cells [
44‐
47], but this, together with the study conducted in parallel by DiGruccio et al [
43], is the first study to compare delta cells with neighbouring alpha and beta cells. Delta cells exert a tonic inhibitory tone over both insulin and glucagon release, as evident from the elevated rates of basal insulin and glucagon release from perfused pancreases in the presence of SSTR inhibitors. Whether and how agonists/antagonists modulate SST signalling will therefore be an important consideration in the design of new antidiabetic drug targets, as well as for our understanding of the endocrine and metabolic control of insulin secretion.
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