1. Possible new treatments for Congenital Hyperinsulinism
Investigating the insulinostatic effects of the somatostatin
analogue, Pasireotide and the RFamide peptides, QRFP43 and 26RFa
Edward S Jones
Faculty of Life Sciences
INTRODUCTION
HYPOTHESIS AND OBJECTIVES
METHODS
Congenital hyperinsulinism (CHI) is a heterogeneous metabolic disorder characterised by insulin excess1. Treatments currently include removal of affected pancreatic tissue and pharmaceutical interventions such as Octreotide, a somatostatin receptor agonist, which suppresses the secretion of a number of hormones, including insulin. Recently, a new somatostatin receptor agonist Pasireotide and a neuropeptide QRFP have shown efficacy in animal studies2,3. Both compounds have insulin-suppressing properties in vivo and may be of use in the treatment of disorders of insulin excess but whether they directly influence insulin secretion from islets of Langerhans has not been investigated.
The aim of this study was to test the hypothesis that the somatostatin receptor agonist Pasireotide and neuropeptide QRFP can modulate insulin secretion from isolated murine islets
Islet Isolation: Pancreatic Islets of Langerhans were isolated from male BALB/c mice using two isolation techniques: ex vivo lobe injection (n=4 isolations) and bile duct cannulation in situ4 (n = 11 isolations).
Measurement of insulin secretion: Murine islets (3 per Eppendorf tube in 300 µl Krebs-Ringer HEPES buffer) were incubated at 37°C in a water bath and treated in triplicate with secretagogue ± peptide agonists (1 hour) as indicated in the Figures. Noradrenaline was used as an established inhibitor of insulin secretion. Supernatants were retained for measurement of insulin content using radioimmunoassay (Figures 3 and 4) or ELISA (Figures 2 and 5). Samples were assayed in duplicate and insulin release is expressed as ng/islet/hour.
Statistical Analysis: Data are presented as mean ± SEM and were analysed using Graph Pad PRISM version 6 software (refer to Figures for details).
The objectives of the study were to:
establish an efficient method for isolating islets from whole murine pancreas via quantitative comparison of two isolation techniques: ex vivo lobe injection and in situ bile duct cannulation
confirm that isolated murine islets respond to insulin secretagogues
elucidate whether exposure to Pasireotide, QRFP43 and 26RFa can inhibit stimulus-dependent insulin secretion
RESULTS
Figure 1: A greater islet yield is obtained using in situ bile duct cannulation compared to pancreatic lobe injection ex vivo
Figure 2: Glucose stimulates insulin secretion from isolated murine islets in a concentration-dependent manner
Figure 3: Pasireotide suppresses glucose-stimulated insulin secretion
Figure 4: Pasireotide does not inhibit arginine-stimulated insulin secretion
Figure 5: Neither QRFP43 nor 26RFa affect basal insulin release but QRFP43 potentiates glucose-stimulated insulin secretion
***p< vs 2mM glucose; # # #p<0.0001; # #p<0.001 vs 20mM glucose. One-way ANOVA with Tukey’s post hoc test (n=3). Pas = Pasireotide
**p<0.01vs 2mM glucose; # # #p<0.0001vs 20mM arginine. NS = not significant. One way ANOVA with Tukey’s post hoc test (n=3)
Islets isolated by bile duct cannulation are glucose responsive and viable. ****p< vs 2mM glucose; one way ANOVA with Tukey’s post hoc test. NS = not significant (n=3).
Lobe injection technique (n=4) versus bile duct cannulation (n=11) ****p<0.0001; unpaired t-test.
****p<0.0001; ++++p<0.001; ####p<0.0001; two way ANOVA with Bonferroni’s post hoc analysis)
SUMMARY AND CONCLUSIONS
Islet isolation
Insulin secretory response
The in situ bile duct cannulation technique results in significantly more islets retrieved per murine pancreas than the ex vivo lobe injection technique (Figure 1).
Isolating murine islets using the bile duct cannulation technique produces viable, glucose- and arginine-responsive islets (Figures 2-5).
Pasireotide significantly reduces glucose-stimulated insulin secretion from murine islets but does not modulate arginine-stimulated insulin secretion (Figure 4). This suggests that the agonist inhibits islet secretory responses to metabolic but not to depolarising stimuli.
QRFP43 and 26RFa do not inhibit basal insulin release but QRFP43 unexpectedly potentiates glucose-stimulated insulin secretion through an unidentified mechanism (Figure 5).
Future work should focus on the effects of Pasireotide on glucagon secretion to determine whether the drug is appropriate for CHI treatment.
Data from this in vitro study showing potentiation of stimulated insulin secretion suggest that the QRFP compounds would not be appropriate as therapeutic agents for CHI.
ex vivo
in situ
Acknowledgments: Thanks to Chris Cook and to my supervisors Dr Karen Cosgrove, Prof. Simon Luckman and Prof. Mark Dunne for their support. Thank you also to the Diabetes Research Group at King’s College London for training and advice on islet isolation.
References: 1. Arnoux et al (2010) Congenital Hyperinsulinism. Early Human Development 86 (5); Arnaldi et al (2010) Pasireotide for the treatment of Cushing's disease. Expert Opinion on Investigational Drugs 19 (7); Egido et al (2007) 26RFa inhibits insulin secretion in the rat pancreas. Peptides 28 (4); Carter et al (2009) A practical guide to rodent islet isolation and assessment. Biological Procedures Online 11 (1); 3-31.