Abstract
This study was designed to determine the effects of 10 and 13 amino acid forms of kisspeptin on dynamic insulin secretion from mammalian islets since it is not clear from published data whether the shorter peptide is stimulatory while the longer peptide inhibits insulin release. Insulin secretion was measured by radioimmunoassay following perifusion of human, pig, rat and mouse isolated islets with kisspeptin-10 or kisspeptin-13 in the presence of 20 mM glucose. Both peptides stimulated rapid, reversible potentiation of glucose-stimulated insulin secretion from islets of all species tested. These data indicate that both kisspeptin-10 and kisspeptin-13, which is an extension of kisspeptin-10 by three amino acids, act directly at islet β-cells of various species to potentiate insulin secretion, and suggest that inhibitory effects reported in earlier studies may reflect differences in experimental protocols.
Introduction
Kisspeptins are a family of peptides encoded by the KISS-1 gene, which have been identified as the endogenous ligands for the G-protein coupled receptor, GPR54.1 The KISS-1 gene encodes a 145-amino acid protein, which is cleaved into various forms including kisspeptin-54, kisspeptin-14, kisspeptin-13 and kisspeptin-10, named with respect to their length in amino acids. Both kisspeptin and GPR54 are expressed within islets of Langerhans,2 suggesting a potential local role for kisspeptin in modulating islet function. There have been only five papers published to date investigating the effect of kisspeptin on insulin secretion, and there is currently no consensus on the role of GPR54 activation in insulin output. Thus, there have been reports of kisspeptin potentiating glucose-induced insulin secretion2-4 inhibiting basal5 and glucose-stimulated6 insulin release, and having no effect in the presence of stimulatory concentrations of glucose.5 The reasons for discrepancies between different studies are not clear, but it is possible that differences in experimental design, including mammalian species and length of kisspeptin used, may account for the conflicting observations. For example, stimulatory effects have been observed with kisspeptin-10,2-4 while the 13 amino acid peptide is associated with inhibition of insulin secretion.5,6 In addition, a variety of experimental protocols, including perfused pancreas, perifused islets and static incubations of islets have been used for measurements of insulin release. We have, therefore, used a standardized perifusion protocol to investigate the effects of kisspeptin-10 and kisspeptin-13 on insulin secretion from isolated mouse, rat, human and piglet islets, to determine whether either mammalian species or kisspeptin amino acid sequence may explain the contradictions in the reported studies.
Results
Increasing the glucose concentration from 2 mM to a maximal stimulatory concentration of 20 mM initiated a biphasic insulin secretory response from mouse (Fig. 1A), rat (Fig. 1B), human (Fig. 1C) and piglet (Fig. 1D) islets, as expected. We have previously shown that the second plateau phase of glucose-induced insulin secretion continues at a similar level in the absence of further treatments for at least 1 h.3 Administration of 1 μM of either kisspeptin-10 or kisspeptin-13 to mouse islets during the stable second phase of glucose-induced secretion resulted in a rapid potentiation of secretion (Fig. 1A). The extent of stimulation of insulin release from mouse islets was similar for both peptides, with approximately 3-fold increases (p < 0.01 vs second phase glucose-induced secretion), which were readily reversible upon removal of the peptides. The basal rate of insulin secretion from islets of all species was comparable, but the magnitude of insulin secretion at 20 mM glucose was approximately 3- to 4-fold lower in rat, human and piglet islets than in mouse islets. As had been observed with mouse islets, insulin secretion from rat, human and piglet islets was rapidly stimulated by addition of either 1 μM kisspeptin-10 or kisspeptin-13 during the second phase of glucose-induced secretion (p < 0.01), although the insulin secretory response to both peptides was more transient in rat islets with a decline in secretion despite the continued presence of the peptides. In addition, whereas the 10 and 13 amino acid kisspeptides stimulated insulin release from mouse, human and piglet islets to an equal extent, the peak response to kisspeptin-10 was significantly greater than that to kisspeptin-13 in rat islets (p < 0.01 at t = 38 min).
Published online:
01 January 2012Figure 1. Kisspeptins reversibly increase insulin secretion from islets isolated from mouse, rat, human and piglet pancreas. Increasing the glucose concentration from 2 mM (0–10 min) to 20 mM (white bar) induced a biphasic stimulation of insulin secretion from mouse (A), rat (B), human (C) and piglet (D) islets prior to kisspeptin (KP) treatment. Exposure to 1 μM kisspeptin-10 (solid symbols) or kisspeptin-13 (open symbols) as shown by the black bar (30–50 min) significantly potentiated the plateau phase of glucose-stimulated insulin secretion from isolated islets of all species tested (p < 0.05, kisspeptin peak insulin release vs. pre-kisspeptin glucose-induced insulin release). Data are means ± SEM, n = 4 for all panels.
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Figure 1. Kisspeptins reversibly increase insulin secretion from islets isolated from mouse, rat, human and piglet pancreas. Increasing the glucose concentration from 2 mM (0–10 min) to 20 mM (white bar) induced a biphasic stimulation of insulin secretion from mouse (A), rat (B), human (C) and piglet (D) islets prior to kisspeptin (KP) treatment. Exposure to 1 μM kisspeptin-10 (solid symbols) or kisspeptin-13 (open symbols) as shown by the black bar (30–50 min) significantly potentiated the plateau phase of glucose-stimulated insulin secretion from isolated islets of all species tested (p < 0.05, kisspeptin peak insulin release vs. pre-kisspeptin glucose-induced insulin release). Data are means ± SEM, n = 4 for all panels.
Discussion
The identification of islet cells expressing kisspeptin and its receptor, GPR54,2 raise the possibility that this peptide has an autocrine or paracrine signaling role to regulate insulin secretion. Studies published to date report that kisspeptin-10, the shortest kisspeptin sequence with biological activity,1 stimulates insulin secretion from mouse islets in vitro2,3 and from rats3 and monkeys4 in vivo, while the longer peptide, kisspeptin-13, inhibits insulin release in vitro from mouse islets5 and rat perfused pancreas.6 All identified forms of kisspeptin, including 10 and 13, bind with similar affinities to GPR54 in all species tested1 and it seems unlikely that different forms of kisspeptin might cause opposite effects on insulin release through activation of GPR54. However, it cannot be ruled out that some forms of kisspeptin can bind to another, currently unidentified, receptor resulting in the reported discrepancies.
To investigate the apparent contradictions within existing studies, the aims of the experiments described in this brief report were 2-fold: to determine whether an additional three amino acids confers an inhibitory mode of action to kisspeptin-10, and to identify whether there are differences between islets of different species in their insulin secretory responses to kisspeptins. A standard perifusion protocol was used to allow dynamic measurement of insulin secretion from isolated islets in the same experiments, using the same buffers supplemented with 1 μM kisspeptin-10 or -13.
The results presented here, showing that both kisspeptin-10 and kisspeptin-13 reversibly potentiate glucose-induced insulin release from mouse, rat, human and piglet islets, indicate that there is no major species difference when using the same methodology. The data are consistent with GPR54 being coupled to stimulatory cascades in β-cells2,3 and do not support the existence of a novel kisspeptin receptor that mediates inhibitory effects of kisspeptin-13. The reasons for inhibition of insulin secretion by kisspeptin-13 observed in two earlier studies5,6 is not, therefore, a consequence of the 13 amino acid peptide acting in a different manner to kisspeptin-10, but most likely reflects differences in experimental protocols. In the perfused rat pancreas experiments, infusion of kisspeptin-13 profoundly inhibited insulin secretion in response to both nutrients and receptor agonists coupled to Gq and Gs,6 suggesting an effect at a late stage in the β-cell exocytotic cascade. This is reminiscent of the inhibitory effects of noradrenaline on insulin secretion7 and it is possible that kisspeptin stimulates noradrenaline release from sympathetic nerve terminals that may be preserved in the perfused pancreas preparation.
While the inhibition of glucose-induced insulin release in response to kisspeptin in perfused rat pancreas may be attributed to differences in methodology, it is more difficult to explain the discrepancies seen in superficially similar studies using isolated mouse islets.2,3,5 The main differences between the contradictory static incubation experiments using mouse islets is that one study used kisspeptin-13,5 while two others used kisspeptin-10,2,3and kisspeptin-13 was reported to exert small inhibitory effects at sub-stimulatory (2.8 mM) glucose while having no significant effect on insulin secretion at a maximal stimulatory (16.7 mM) glucose concentration.5 The data presented here show that both peptides produce similar stimulatory secretory profiles in mouse islets at 20 mM glucose. Elevations in insulin secretion may not be visible over 1 h in static incubations and accumulation of potentially inhibitory agents such as somatostatin, gamma-aminobutyric acid or peptide YY could repress insulin secretion. The rapid flow in the current perifusion experiments minimizes indirect inhibition of insulin output by other secreted islet hormones and the minute by minute sample collection in perifusion experiments allows sensitive monitoring of the profile of dynamic insulin release. There is relatively little in vivo data regarding the effects of kisspeptin on insulin release, but studies have shown that administration of kisspeptin in both monkeys and rats results in significant stimulation of insulin release.3,4 Although further in vivo work is required to fully characterize the physiological role of the islet kisspeptin system, these studies provide the clearest indication toward the physiological role of kisspeptin on insulin release and suggest that the results obtained in the present study are not purely a pharmacological in vitro phenomenon.
In summary, our results indicate that, at least locally within islets, kisspeptin-10 and kisspeptin-13 act at the same receptor, resulting in stimulation of insulin secretion. Thus, while the discrepancies between published studies reporting the effects of kisspeptin on insulin secretion are still not fully understood, these differences are not due to either kisspeptin length or species used for islet isolation.
Materials and Methods
Materials
Mouse kisspeptin-10 and kisspeptin-13 were synthesized by Alta Biosciences. Collagenase XI was from Sigma-Aldrich, and culture media and fetal calf serum were from Invitrogen.
Islet isolation
Islets were isolated from ICR mice, Sprague-Dawley rats, neonatal pigs (20 ± 5 d) and heart-beating organ donors by collagenase digestion of the exocrine pancreas,8 and maintained in culture for at least 48 h at 37°C (95%/5% air/CO2) before experimental use.
Insulin secretion in vitro
Groups of 40 islets were perifused at 37°C9 with a physiological salt solution,10 and insulin content was determined by radioimmunoassay.11
Statistical analysis
Secretion data are expressed as means ± SEM. Statistical analyses were performed using Student’s t-tests and peak insulin release during kisspeptin administration was compared with glucose-induced insulin release during the plateau phase, prior to kisspeptin administration. p < 0.05 was considered statistically significant.
Acknowledgments
This work was supported by the Diabetes Research & Wellness Foundation and Diabetes UK (06/0003316 and 07/0003510). JB is a DRWF Research Fellow. We are grateful to the relatives of organ donors for human pancreata for islet isolation.
No potential conflicts of interest were disclosed.
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