Effects of 2 Novel PYY(1-36) Analogues, (P³L³¹P³⁴)PYY(1-36) and PYY(1-36)(Lys¹²PAL), on Pancreatic Beta-Cell Function, Growth, and Survival

Abstract

Recent studies have identified a beneficial role for peptide tyrosine tyrosine (PYY) on pancreatic beta-cell function and survival. These effects are linked to the activation of neuropeptide Y1 receptors (NPYR1s) by PYY(1-36). However, PYY(1-36) is subject to rapid degradation by dipeptidyl peptidase-4 (DPP-4), resulting is the loss of NPYR1 activity. Therefore, the aim of this study was to develop 2 enzymatically stable PYY(1-36) analogues, namely, (P 3 L 31 P 34 )PYY(1-36) and PYY(1-36)(Lys 12 PAL), with further structural modifications to enhance NPYR1 specificity. As expected, (P 3 L 31 P 34 )PYY(1-36) was fully resistant to DPP-4-mediated degradation in vitro, whereas PYY(1-36) and PYY(1-36)(Lys 12 PAL) were both liable to DPP-4 breakdown. PYY(1-36) and (P 3 L 31 P 34 )PYY(1-36) induced significant reductions in glucose-stimulated insulin secretion (GSIS) from BRIN BD11 cells, but only PYY(1-36) diminished alanine-stimulated insulin secretion. In contrast, PYY(1-36)(Lys 12 PAL) had no impact on GSIS or alanine-induced insulin release. All 3 PYY peptides significantly enhanced proliferation in BRIN BD11 and 1.1B4 beta-cell lines, albeit only at the highest concentration examined, 10 -6 M, for (P 3 L 31 P 34 )PYY(1-36) and PYY(1-36)(Lys 12 PAL) in BRIN BD11 cells. Regarding the protection of beta-cells against cytokine-induced apoptosis, PYY(1-36) induced clear protective effects. Both (P 3 L 31 P 34 )PYY(1-36) and PYY(1-36)(Lys 12 PAL) offered some protection against apoptosis in BRIN BD11 cells, but were significantly less efficacious than PYY(1-36). Similarly, in 1.1B4 cells, both PYY analogues (10 -6 M) protected against cytokine-induced apoptosis, but (P 3 L 31 P 34 )PYY(1-36) was significantly less effective than PYY(1-36). All 3 PYY peptides had no impact on refeeding in overnight fasted mice. These data underline the beta-cell benefits of PYY(1-36) and highlight the challenges of synthesising stable, bioactive, NPYR1-specific, PYY(1-36) analogues.

Peptide YY (1–36) peptides from phylogenetically ancient fish targeting mammalian neuropeptide Y1 receptors demonstrate potent effects on pancreatic β‐cell function, growth and survival

“…Reaction mixes were separated by RP‐HPLC using a (250 × 4.6 mm) Phenomenex C‐18 analytical column, with absorbance monitored at 214 nm using a SpectraSystem UV2000 detector (Thermoquest, Manchester, UK). Defined HPLC peaks were collected and identified via MALDI‐TOF mass spectrometry (MS) on a Voyager‐DE Biospectrometer (PerSeptive BioSystems, Framingham, Massachusetts), to corroborate peptide stability, as described previously …”

“…Following removal of pre‐incubation buffer, 1 mL of KRB test solution, containing either 5.6 or 16.7 mM glucose with PYY (1–36) test peptides (10 −12 – 10 −6 M) was added. In a second series of experiments, the insulin secretory effects of PYY (1–36) peptides were determined after incubation in the presence of alanine (10 mM) . For all experiments, following a 20‐minute incubation period, supernatant was collected and stored at –20°C until determination of insulin concentrations by a fully characterized dextran‐coated charcoal radioimmunoassay …”

“…All slides were viewed using a fluorescent microscope (Olympus System Microscope, model BX51; Olympus, Southend‐on‐Sea, UK) and photographed by DP70 camera adapter system. Proliferation/TUNEL positive frequency was determined using the cell‐counter function on ImageJ Software and expressed as percentage of total cells analysed, as described previously in studies from our laboratory …”

“…Consequently, N‐terminally stabilized PYY (1–36) peptide analogues, resistant to DPP‐4 degradation to maintain NPYR1 activity, are of particular interest. A recent study has employed available PYY/NPYR1 structure/function knowledge in an attempt to generate enzymatically stable, NPYR1‐specific, PYY (1–36) sequences . However, the bioactivity of these chemically engineered PYY (1–36) peptides was less impressive than anticipated .…”

“…A recent study has employed available PYY/NPYR1 structure/function knowledge in an attempt to generate enzymatically stable, NPYR1‐specific, PYY (1–36) sequences . However, the bioactivity of these chemically engineered PYY (1–36) peptides was less impressive than anticipated . Consequently, there is still an important need to develop stabilized PYY (1–36) peptides, with NPYR1 specificity, as a possible new and effective class of antidiabetic therapeutics.…”