Elsevier

Metabolism

Volume 111, October 2020, 154339
Metabolism

Basic Science
Generation and characterisation of C-terminally stabilised PYY molecules with potential in vivo NPYR2 activity

https://doi.org/10.1016/j.metabol.2020.154339Get rights and content

Highlights

  • Activation of neuropeptide Y2 receptors (NPYR2's) induces anti-obesity effects

  • We have created Y2 agonists with C-terminal enzymatic liability or stability, based on the structure of sea lamprey PYY

  • Twice daily administration of either Y2 agonist to high fat fed mice resulted in significant metabolic improvements

  • Interestingly, therapeutic benefits of imparting C-terminal stability were not overly apparent

Abstract

Background

Activation of neuropeptide Y2 receptors (NPYR2) by the N-terminally truncated, dipeptidyl peptidase-4 (DPP-4) generated, Peptide YY (PYY) metabolite, namely PYY(3–36), results in satiating actions. However, PYY(3–36) is also subject to C-terminal enzymatic cleavage, which annuls anorectic effects.

Methods

Substitution of l-Arg35 with d-Arg35 in the DPP-4 stable sea lamprey PYY(1–36) peptide imparts full C-terminal stability. In the current study, we have taken this molecule and introduced DPP-4 susceptibility by Iso3 substitution.

Results

As expected, [Iso3]sea lamprey PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36) were N-terminally degraded to respective PYY(3–36) metabolites in plasma. Only [Iso3](d-Arg35)sea lamprey PYY(1–36) was C-terminally stable. Both peptides possessed similar insulinostatic and anti-apoptotic biological actions to native PYY(1–36) in beta-cells. Unlike native PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36), [Iso3]sea lamprey PYY(1–36) displayed some proliferative actions in Npyr1 knockout beta-cells. In addition, [Iso3]sea lamprey PYY(1–36) induced more rapid NPYR2-dependent appetite suppressive effects in mice than its C-terminally stable counterpart. Twice daily administration of either peptide to high fat fed (HFF) mice resulted in significant body weight reduction and improvements in circulating triglyceride levels. [Iso3]sea lamprey PYY(1–36) treatment also prevented elevations in glucagon. Both peptides, and especially [Iso3]sea lamprey PYY(1–36), improved glucose tolerance. The treatment interventions also partially reversed the deleterious effects of sustained high fat feeding on pancreatic islet morphology.

Conclusion

The present study confirms that sustained NPYR2 receptor activation by [Iso3](d-Arg35)sea lamprey induced significant weight lowering actions. However, identifiable benefits of this peptide over [Iso3]sea lamprey PYY(1–36), which was not protected against C-terminal degradation, were not pronounced.

Introduction

Peptide Tyrosine Tyrosine (PYY) is a 36 amino acid, gut-derived hormone secreted postprandially from enteroendocrine L-cells [1]. Upon release, PYY is subject to rapid degradation by the ubiquitous enzyme dipeptidyl peptidase-4 (DPP-4), resulting in removal of the N-terminal dipeptide, Tyr1-Pro2 [2]. Whilst the parent peptide, PYY(1–36), is a full agonist at each of the identified neuropeptide Y receptors (NPYR), namely NPYR1, NPYR2, NPYR4 and NPYR5 [3], PYY(3–36) is highly specific for NPYR2 [4]. Since activation of hypothalamic NPYR2 leads to appetite suppression in various animal models [5,6], as well as normal and obese humans [5,[7], [8], [9], [10]], the potential application of PYY(3–36) based therapies for obesity has been investigated [11,12].

Despite this early enthusiasm, PYY(3–36) is yet to be clinically exploited. One potential explanation could be the adverse effects that occur following administration of PYY(3–36) in humans, which include nausea, sweating and abdominal discomfort [13]. However, such effects are dose-dependent [13], suggesting they could be overcome by utilisation of appropriate dosing regimens as is the case for clinical use of GLP-1 mimetics [[14], [15], [16]]. An additional explanation for the lack of clinical uptake may be linked to recent documentation of C-terminally directed enzymatic cleavage of PYY peptides. As such, it has been revealed that PYY(3–36) is rapidly degraded to PYY (3–34) [17] which exhibits no anorexigenic actions [17,18] and is postulated to be the most abundant PYY peptide in the circulation [18]. Similar C-terminal truncation and loss of NPYR activity, has been reported for the parent peptide, PYY(1–36) [19]. Together, these observations confirm the importance of C-terminal integrity for the bioactivity of PYY molecules [20].

C-terminally stable forms of PYY(3–36) have been documented [21], but biological and potential therapeutic efficacy of such NPYR2 specific peptides has not been established. More recent studies have characterised, and fully investigated the bioactivity, of a C-terminally stabilised, mammalian NPYR1 agonist, based on the amino acid sequence of sea lamprey PYY(1–36) that possesses inherent resistance to DPP-4 [[22], [23], [24]]. C-terminal stability of sea lamprey PYY(1–36) was introduced by substitution of l-Arg35 with its enantiomer d-Arg35 [24]. Thus, (d-Arg35)sea lamprey PYY(1–36) is a recently described, fully characterised, N- and C-terminally stabilised PYY peptide form [24].

The present study has progressed the work, using this peptide template, to generate viable PYY(3–36) forms, with proposed NPYR2 activity and potential anti-obesity actions. Thus, Pro3 in sea lamprey PYY(1–36) was substituted by Iso3, the naturally occurring residue in human PYY, to encourage DPP-4 susceptibility, leading to generation of [Iso3]sea lamprey PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36). The rationale being that DPP-4 cleavage of these PYY analogues would generate specific NPYR2 agonists [2]. Moreover, transient activation of NPYR1, by the parent peptides in vivo before cleavage, could be beneficial at the level of the endocrine pancreas, as reported previously [[22], [23], [24]]. Although synthesis of [Iso3]sea lamprey PYY(3–36) would also potentially lead to generation of a specific NPYR2 agonist, any NPYR1 benefits of the parent peptide would be lost. We therefore decided not to investigate this avenue within the current study. Initially, DPP-4 stability of [Iso3]sea lamprey PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36) was demonstrated. Further studies examined effects on pancreatic beta-cell function and survival in vitro, as well as NPYR1 and NPYR2 specificity. The acute impact of the PYY peptides on satiety was then investigated in vivo. Finally, preclinical therapeutic potential of [Iso3]sea lamprey PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36) was investigated in obese high fat fed (HFF) mice.

Section snippets

Peptides

All peptides were supplied by Synpeptide Ltd. (Shanghai, China) in excess of 95% purity and characterised in-house using HPLC with MALDI-TOF (Table 1), as described previously [24].

Plasma degradation

[Iso3]sea lamprey and [Iso3](d-Arg35)sea lamprey PYY(1–36) peptides (50 μg) were incubated at 37 °C on a plate-shaker in 50 mM triethanolamine/HCl (pH 7.8) with 5 μl non-fasted C57BL/6 murine plasma for 0 and 4 h, as described previously [19]. In an additional experiment, [Iso3]sea lamprey PYY(1–36) was incubated as

In vitro plasma stability

Incubation of [Iso3]sea lamprey PYY(1–36) or [Iso3](d-Arg35)sea lamprey PYY(1–36) with murine plasma resulted in the generation of respective PYY(3–36) metabolites (Fig. 1A,B, Table 1). However, when incubated concomitantly with the DPP-4 inhibitor, sitagliptin, [Iso3](d-Arg35)sea lamprey PYY(1–36) remained fully intact (data not shown), whereas [Iso3]sea lamprey PYY(1–36) was degraded to [Iso3]sea lamprey PYY (1–34) (Fig. 1C).

Effects of sea lamprey PYY(1–36) analogues on insulin secretion from BRIN BD11 beta-cells

Native PYY(1–36), sea lamprey PYY(1–36) and both [Iso3] substituted

Discussion

In the present investigation, (d-Arg35)sea lamprey PYY(1–36) [24] was rendered DPP-4 susceptible by Iso3 for Pro3 substitution, as confirmed in vitro, generating the C-terminally stable NPYR2 agonist, [Iso3](d-Arg35)sea lamprey PYY(3–36). Thus, the parent peptide, [Iso3](d-Arg35)sea lamprey PYY(1–36), will exert benefits at the level of the endocrine pancreas through positive modulation of NPYR1 [[22], [23], [24]], whereas the generated metabolite, [Iso3](d-Arg35)sea lamprey PYY(3–36), will

CRediT authorship contribution statement

Ryan A. Lafferty:Conceptualization, Methodology, Validation, Data curation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization.Neil Tanday:Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing.Peter R. Flatt:Conceptualization, Methodology, Validation, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.Nigel Irwin:Conceptualization,

Declaration of competing interest

PRF and NI are named on patents filed by the University of Ulster for exploitation of peptide therapeutics.

Acknowledgment

This work was supported by a PhD studentship (awarded to RAL) from the Department for the Economy (DfE) Northern Ireland, Invest Northern Ireland Proof of Concept funding and University of Ulster strategic research funding.

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