Srinivasan D1, Ojo OO1, Flatt PR1, Conlon JM2, Abdel-Wahab YHA1 [Lys-3] Substitution of peptide Alyteserin-2a from skin secretions of the midwife toad Alytes obstetricans enhances insulin-releasing activity in vitro. Srinivasan D1, Ojo OO1, Flatt PR1, Conlon JM2, Abdel-Wahab YHA1 1School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, BT52 1SA, UK. 2Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 , UAE Corresponding author: Srinivasan-d1@email.ulster.ac.uk INTRODUCTION Figure 2: Effects of [Lys-3]Alyteserin-2a on insulin release from BRIN-BD 11 cells in the presence of known modulators of insulin release (A) and absence of extracellular calcium (B) Diabetes mellitus (DM) is characterised by hyperglycaemia. It is estimated that 552 million people worldwide and 4 million people in the UK will be diagnosed with diabetes in 2030. The drawbacks associated with current drugs such as side effects (sulfonylureas and TZDs), high cost (glinides) and short-term action (incretins) necessitate intense research into finding new drugs for diabetes treatment. Skin secretions of amphibians are shown to be rich in antimicrobial peptides. Research into the anti-diabetic use of the peptides from animal secretions took centre stage after the discovery of Exendin-4. Exendin-4, a peptide homologous to GLP-1, was isolated from saliva of lizard, Heloderma suspectum and has since been found to be useful in diabetes treatment. Alytes obstetricans, Commonly called the midwife toad, are native to regions of Europe and Africa. Peptides alyteserin-1 and alyteserin-2, isolated from skin secretions of A. obstetricans are shown to posses antimicrobial activities (Conlon et al., 2009). Previous studies in our laboratory have elucidated the insulinotropic properties of alyteserin-2a. Reports suggest that synthetic analogues of peptides, with increased cationicity and hydrophobicity, have more potent insulin releasing activities compared to native peptides (Abdel-Wahab et al., 2008) Values are Mean ± SEM (n=8). *P<0.05, **P<0.01, ***P<0.001 compared to 5.6mM glucose alone in the absence of the peptide (A) or presence of extracellular calcium (B). +P<0.05, ++P<0.01, +++P<0.001 compared to 5.6mM glucose in the presence of the peptide. ΔP<0.05, ΔΔP<0.01, ΔΔΔP<0.001 compared to respective incubation in the absence of the peptide. Stimulatory effects of [Lys-3] Alyteserin-2a (10-6 M) were inhibited upon incubation with 50µM verapamil and 300µM diazoxide. At 5.6mM glucose and 10-6 M [Lys-3] Alyteserin-2a, incubation with 30mM KCl, 200µM IBMX and 200µM tolbutamide resulted in increased insulin release. In the absence of extracellular calcium, stimulation of insulin release by [Lys-3] alyteserin-2a (10-6M) was reduced by 33.6% (P< 0.001), compared to the stimulation of insulin release in the presence of extracellular calcium at the same concentration (Figure 2). L G S N I K T A L G S N I K T A Fig. 3: Effects of [Lys-3] alyteserin-2a on intracellular calcium (A) and membrane potential (B) in BRIN-BD 11 cells expressed as RFU and area under the curve. NH2 NH2 Alyteserin-2a [Lys-3] Alyteserin-2a OBJECTIVES The current study investigated the insulin-releasing properties and cytotoxicity of a synthetic analogue of alyteserin-2a with increased cationicity, [Lys-3] Alyteserin-2a and the possible mechanism of action using BRIN-BD11 clonal pancreatic β cell lines. METHODS Assessment of Beta cell function: Acute test: Acute insulin-release studies were performed using Krebs Ringer Bicarbonate buffer (KRBB) supplemented with 5.6mM glucose in the presence of native alyteserin-2a and [Lys-3] alyteserin-2a at different concentrations (0-3×10-6 M) and known modulators of insulin release. Radioimmunoassay: Insulin released during the acute exposure to test solutions was assayed using radioimmunoassay, which works by the principle of competitive binding between insulin in the sample and radio-labelled insulin. Assessment of Cell Viability: LDH levels were used as an indicator to assess the cytotoxic effects of the peptides on the cells. LDH assay was performed using Cytotox-96 non-radioactive cytotoxicity assay kit (Molecular devices, Sunnyvale, USA). Intracellular calcium and membrane potential assays: Intracellular calcium levels and membrane potential were determined by fluorometric assays using FlexStation micro plate reader (Molecular devices, Sunnyvale, USA). Values are Mean ± SEM (n=6). ***P<0.001, **P<0.01 *P<0.05 compared to 5.6mM glucose alone At 5.6mM glucose, Incubation of BRIN-BD11 cells with [Lys-3] alyteserin-2a (10-6M) caused an increase in intracellular calcium levels in BRIN-BD11 cells (2.5 fold increase, P<0.001). A significant increase in membrane potential was observed, owing to the depolarisation of the membrane by [Lys-3] alyteserin-2a (0.5 fold increase, P<0.01) (Figure 3). DISCUSSION Alyteserin-2a and [Lys-3] alyteserin-2a stimulated insulin release from BRIN-BD11 cells in a dose-dependent manner, at concentrations ranging from (10-9-3x10-6M). [Lys-3] alyteserin-2a was more potent in stimulating insulin release at lower concentrations, compared to alyteserin-2a. Inhibition of insulin stimulation by verapamil and diazoxide suggests that [Lys-3] alyteserin-2a might be acting through the KATP channel-dependent pathway. Increase in intracellular calcium levels and membrane potential support this finding. Enhancement of insulin release by IBMX and KCl in the presence of the synthetic analogue suggests an involvement of secondary messenger pathway. RESULTS Figure 1: Comparative dose dependent effects of alyteserin-2a and [Lys-3] alyteserin-2a on insulin release (A) and LDH release (B) from BRIN BD 11 cells. CONCLUSION Our study demonstrates that synthetic [Lys-3] Alyteserin-2a is more potent than alyteserin-2a from Alytes obstetricans in stimulating insulin release in vitro and identifies the potential of [Lys-3] Alyteserin-2a for development into a new antidiabetic agent. Values are Mean ± SEM with n =6 for insulin and n=3 for LDH. ***P<0.001, **P<0.01, *P<0.05 compared to 5.6mM glucose alone. +++P<0.001, ++P<0.01, +P<0.05 compared to respective concentration of alyteserin-2a. REFERENCES Conlon JM, Demandt A, Nielsen PF, Leprince J, Vaudry H and Woodhams DC 2009 The alyteserins: two families of antimicrobial peptides from the skin secretions of the midwife toad Alytes obstetricans (Alytidae). Peptides 30 1069-73 Abdel-Wahab YHA, Power GJ, Ng MT, Flatt PR and Conlon, JM 2008 Insulin-releasing properties of the frog skin peptide pseudin-2 and its [Lys18]-substituted analogue. Biol. Chem., 389(2) 143-148. At 5.6mM glucose, Alyteserin-2a and [Lys-3] Alyteserin-2a stimulated insulin release in a dose-dependent manner, without beta-cell cytotoxicity. Though Alyteserin-2a and its [Lys-3] substituted analogue produced a similar maximum stimulation of insulin release at 5.6mM glucose (~3.1 fold, 3x10-6M, P<0.001), [Lys-3] Alyteserin-2a was significantly more potent at lower concentrations. At 5.6mM glucose, [Lys-3] alyteserin-2a (1nM) produced a 34.3% increase in insulin release compared to 6.9% increase by alyteserin-2a at the same concentration (Figure 1). ACKNOWLEDGEMENT The research project was funded by Vice Chancellor’s Research Scholarship awarded to Mr Dinesh Srinivasan.