Basic and clinical science posters: Insulinotropic agents and enteroinsular hormones

Comparing the efficacy of novel apelin-13 analogues vs incretin mimetics in treating streptozotocin-induced diabetes in mice

FPM O'Harte, V Parthsarathy, PR Flatt

The SAAD Centre for Pharmacy and Diabetes, University of Ulster, Coleraine, UK

Aims Our previous research showed promising in vivo antidiabetic benefits of apelin-13 peptide analogues in diet-induced obese and diabetic db/db mice. Here, the efficacy of (pGlu)apelin-13-amide and its acylated analogue (pGlu)(Lys⁸GluPAL)apelin-13-amide, were compared with exendin-4 and liraglutide, following chronic (21 days) administration to streptozotocin-induced (STZ) diabetic mice.

Methods Five groups of male NIH-Swiss mice (n=8) received STZ to induce hyperglycaemia (20 ± 2mM blood glucose). Mice were then given twice daily (09:00 and 17:00 h) i.p. injections of saline vehicle, (pGlu)apelin-13-amide, (pGlu)(Lys⁸GluPAL)apelin-13-amide, exendin-4(1–39) or liraglutide (25nmol/kg bodyweight) for 21 days. Control NIH-Swiss mice received twice-daily saline injections.

Results No changes in bodyweight or food intake were observed with any peptide treatments. Apelin analogues and incretin mimetics showed sustained improved glycaemic control (p < 0.05–p < 0.001, from day 6–21) and reduced non-fasting circulating insulin vs saline-treated control mice. Exendin-4 improved glucose tolerance (ipGTT) in STZ-induced diabetic mice (p < 0.05) vs STZ-induced saline-treated controls. After 21 days, both apelin analogues and incretin mimetics showed reduced LDL-cholesterol and elevated HDL-cholesterol (p < 0.05–p < 0.001) in STZ-induced diabetic mice vs saline-treated controls. Apelin analogues and incretin treatments failed to significantly reduce HbA1c after 21 days. All STZ-induced diabetic mice showed a reduced terminal % body fat mass and increased % lean mass compared to lean controls, following terminal DEXA analysis. Total pancreatic insulin content was significantly reduced (55–62%, p < 0.001) in all STZ-induced diabetic mice regardless of treatment.

Summary In conclusion, longer term administration of apelin-13 analogues was as effective as incretin mimetics in counteracting diabetes in STZ-induced diabetic mice.

Acknowledgement Diabetes Research Group

Identification of multiple pancreatic and extra-pancreatic pathways underlying the glucose lowering actions of Annona squamosa leaf in type 2 diabetes

YHA Abdel-Wahab, P Ansari, PR Flatt

School of Biomedical Sciences, Ulster University, Coleraine, UK

Aims Antidiabetic effects of Annona squamosa leaf have been documented but the underlying mechanisms are not well understood. This study investigated the mechanisms of action together with isolation of active compounds.

Methods Effects of hot water extract of Annona squamosa(HWAS) on insulin release, membrane potential and intracellular calcium ([Ca²⁺]i) were determined using BRIN-BD11 cells and isolated mouse pancreatic islets. Glucose uptake by 3T3 differentiated-adipocyte cells, starch digestion, protein glycation and DPP-IV enzyme activity were determined in vitro. Effects of 9 days oral treatment of HWAS (250mg/5ml/kg) on glucose homeostasis were assessed in high-fat-fed diabetic rats. Active compounds were characterised by HPLC, LCMS and NMR.

Results HWAS (40–5000μg/ml) significantly (p < 0.05–p < 0.001) stimulated insulin release by 1.5- to 3.2-fold in concentration-dependent manner from BRIN-BD11 cells and isolated islets which were comparable to GLP-1. HWAS induced membrane depolarisation (4.5-fold,) and increased [Ca²⁺]i (24-fold) with insulinotropic action significantly inhibited (p < 0.01) by verapamil (38%), diazoxide (37%) and calcium free conditions (64%). Secretory effects of HWAS were further potentiated 1.2- to 1.5-fold (p < 0.01) by IBMX (200μM) tolbutamide (200μM) and KCl (30mM). HWAS significantly increased cellular glucose uptake (1.2- to 1.8-fold, p < 0.05–0.001), inhibited starch digestion, protein glycation and DPP-IV activity (7.5–50%, p < 0.05–p < 0.001). Treatment of rats with HWAS improved blood glucose, oral glucose tolerance, plasma and pancreatic insulin content and inhibited DPP-IV enzyme activity. The most potent isolated compound with insulin releasing activities of molecular mass 610.3Da, was structurally identified as rutin (C27H30O16).

Conclusion HWAS improves glucose homeostasis via multiple pathways in type 2 diabetes representing a novel source of antidiabetic agents.

Assessment of insulin-releasing actions and cytotoxicity of caerulein precursor fragment-SE3 (CPF-SE3) in clonal pancreatic beta cells, BRIN-BD11

OO Ojo¹, JN Edeani¹, AA Falobi¹, AB Falana¹, YHA Adbel-Wahab², PR Flatt²

¹School of Sciences, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK, ²School of Biomedical Sciences, Ulster University, Coleraine, UK

Aim Peptides belonging to the caerulein family have been reported to exhibit insulin-release stimulatory effects. These include analogues of caerulein precursor fragments isolated from the skin secretion of the frog Silurana epitropicalis. However, mechanism underlying these actions is poorly understood. This study assessed insulin-releasing actions of CPF-SE3 and investigated its mechanisms of actions.

Methods Insulin-releasing actions of CPF-SE3 were investigated in buffers containing, graded concentrations of the peptide (0–3μM), increasing glucose concentrations or CPF-SE3 (1μM) with modulators of insulin secretion. Insulin concentrations were measured by ELISA. Cytotoxicity and cell viability were examined in peptide-treated cells. Insulin-releasing effects were also investigated in the absence of extracellular calcium.

Results CPF-SE3 stimulated insulin release from BRIN-BD11 cells in a concentration-dependent manner (1.4- to 3.2-fold, 3nM–3μM, p < 0.001–0.05). The stimulatory effects of CPF-SE3 reduced in the presence of diazoxide (58%, p < 0.001), verapamil 52%, p < 0.01) and in the incubation performed in the absence of extracellular calcium (50%, p < 0.001). Enhanced stimulatory effect was observed in the presence of tolbutamide (200μM, 1.9-fold, p < 0.05) and IBMX (200mM, 2-fold, p < 0.01). Insulinotropic effects of CPF-SE3 increased by 2.2-fold (p < 0.001) and 1.1-fold (p < 0.05) as glucose concentration increased from 1.1mM to 5.6mM and from 5.6mM to 16.7mM. These actions are not associated with reduced cell viability and cytotoxicity.

Conclusions These results suggest that the insulin-releasing effects of CPF-SE3 may involve the KATP-dependent pathway.

Acknowledgement Diabetes Research Group

Investigation of the insulin-releasing actions of aqueous extracts of Ocimum gratissimum

AA Falobi, S Heer, AB Falana, OO Ojo

School of Sciences, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK

Aim Ocimum gratissimum (clove basil) is a widely consumed vegetable in many parts of the world. Antidiabetic actions of extracts of the plant have been reported. However, mechanisms underlying the antidiabetic activity of extracts of the plant remains poorly understood. This study investigated insulinotropic actions, cytotoxic effects and mechanisms involved in the insulin-releasing actions of aqueous extracts of O. gratissimum.

Methods Pancreatic beta cells, BRIN-BD11, were incubated with increasing glucose concentration, extract alone (0–1000μg/ml) or extract (100μg/ml) in the presence or absence of modulators of insulin secretion. Insulin concentrations were measured by ELISA and LDH assay was used to assess cytotoxicity. Viability of treated cells was assessed by MTT assay.

Results O. gratissimum extracts stimulated insulin release from BRIN-BD11 cells in a concentration dependent manner at concentrations ≥10μg/ml (1.4- to 2.5-fold, p < 0.01–0.05). Insulin-releasing effects of O. gratissimum extract were not associated with cytotoxicity at all concentrations tested. Viability reduced by 47% (p < 0.001) in cells treated with 1,000μg/ml extract but not at other concentrations tested. Insulinotropic effects of the extract increased by 2.0-fold (p < 0.001) and 1.2-fold (p < 0.05) as glucose concentration increased from 1.1mM to 5.6mM and from 5.6mM to 16.7mM. Insulin-releasing effects were significantly inhibited in the presence of the KATP channel activator diazoxide (46%, p < 0.001), the L-type calcium channel blocker verapamil (66%, p < 0.001) and in the absence of extracellular calcium (58%, p < 0.001).

Conclusions These results confirm that antidiabetic actions of O. gratissimum extracts involve insulin secretion via the KATP-dependent pathway of insulin secretion.

Acknowledgement Diabetes Research Group

A pseudohexapeptide xenin analogue augments the metabolic benefits of sitagliptin in high-fat-fed mice

VA Gault¹, SL Craig¹, G Hamscher², S McClean¹, N Irwin¹

¹School of Biomedical Sciences, Ulster University, Coleraine, UK, ²Institute of Food Chemistry and Food Biotechnology, Justus Liebig University, Giessen, Germany

Aims The pseudohexapeptide xenin analogue, Ψ-xenin-6, is an enzymatically stable and biologically active form of the gut hormone xenin-25. Ψ-Xenin-6 potentiates the bioactivity of the incretin hormone GIP, suggesting it could enhance the antidiabetic utility of dipeptidyl peptidase-4 (DDP-4) inhibitor drugs.

Methods Studies were conducted in male C57BL/6 mice (n=8; 12 weeks old) given free access to high-fat (45%) diet for 4 weeks. During this period, mice received streptozotocin (STZ) injection (50mg/kg) on weeks 2 and 3. At week 4, mice received oral sitagliptin once daily (50mg/kg), intraperitoneal Ψ-xenin-6 twice daily (25nmol/kg) or a combination of both compounds for 18 days. Body weight, glucose and insulin were measured regularly. Glucose (18mmol/kg), pyruvate (2g/kg) and insulin (50U/kg) tolerance tests were conducted at the end of the treatment period. Pancreatic islet architecture was also assessed.

Results Administration of sitagliptin or Ψ-xenin-6 alone to high-fat-fed mice for 18 days resulted in anticipated benefits including, reduced body weight (p < 0.05), increased circulating and pancreatic insulin (p < 0.001), improved glucose tolerance (p < 0.01) and returning pancreatic islet architecture to that observed in lean control mice. Combined sitagliptin and Ψ-xenin-6 therapy was associated with additional benefits. In these mice, more pronounced (p < 0.01) and rapid benefits on circulating glucose and insulin as well as additional improvements (p < 0.01) in attenuating gluconeogenesis were observed, with favourable effects on pancreatic islet architecture also more apparent.

Conclusion Ψ-Xenin-6 augments the antidiabetic benefits of sitagliptin, representing a potential therapeutic option for type 2 diabetes.

Sea lamprey PYY, an enzymatically stable piscine-derived PYY(1-36) peptide, exerts antidiabetic benefits in streptozotocin-induced diabetic mice

N Irwin¹, RA Lafferty², N Tanday², PR Flatt²

¹School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, UK, ²School of Biomedical Sciences, Ulster University, Coleraine, UK

Aims Sustained activation of neuropeptide Y1 receptors (NPYR1s) induces benefits on pancreatic beta cells. In this regard, we have identified PYY(1–36) sequences from ancient fish with inherent NPYR1 specificity. As such, preclinical antidiabetic efficacy of sustained administration of Petromyzon marinus (sea lamprey) or Scaphirhynchus albus (sturgeon) PYY(1–36) was investigated in diabetic mice.

Methods Diabetes was induced in male NIH Swiss mice (n=8; 12 weeks old) through low-dose streptozotocin (STZ) injection (50mg/kg) for five consecutive days. Mice then received twice daily injections of saline control, sturgeon or sea lamprey PYY(1-36) (25nmol/kg) for 21 days. Body weight, glucose and insulin were measured regularly. Glucose (18mmol/kg) and insulin (50U/kg) tolerance tests were conducted on day 18. Terminal analysis included assessment of pancreatic hormone content and islet architecture.

Results Characteristic body weight reductions and elevated circulating glucose were significantly (p < 0.05–p < 0.01) countered by both piscine-derived PYY(1–36) treatments in STZ-diabetic mice. Corresponding circulating plasma insulin was elevated (p < 0.001) by sea lamprey PYY(1–36). Sea lamprey PYY(1–36) also improved (p < 0.05–p < 0.01) glucose tolerance, glucose-induced insulin secretion and the glucose-lowering action of exogenous insulin. Pancreatic insulin content was elevated (p < 0.05–p < 0.01) in both sea lamprey and sturgeon PYY(1–36) treated mice. Interestingly, sea lamprey treated mice had decreased overall islet area, but increased (p < 0.01) % beta cell and reduced (p < 0.001) % alpha cell areas. Furthermore, proliferating and apoptotic beta cells were significantly (p < 0.001) increased and decreased, respectively, in these mice.

Conclusion Sea lamprey PYY(1-36) induced notable metabolic benefits in STZ-diabetic mice, emphasising that NPYR1 modulators could represent an effective new treatment option for diabetes.

Chronic administration of a long-acting glucagon analogue results in enhanced pancreatic islet function in a diet-induced obesity murine model

K Suba¹, Y Patel¹, A Martin Alonso¹, M Ukwuoma¹, V Kalogianni¹, I Leclerc¹, B Owen¹, GA Rutter², SR Bloom¹, V Salem¹

¹Section of Endocrinology and Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital Campus, London, UK, ²Section of Cell Biology, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital Campus, London, UK

Refer to Oral number A64.