Characterisation of the breakdown of Pancreatic Polypeptide (PP) in vitro and in vivo by the enzymes dipeptidyl peptidase-IV (DPPIV) and neprilysin (24.11)

Jordan Baxter, James Minnion, Joyceline Cuenco-Shillito, Mohammad Ghatei, Steve Bloom. Imperial College, London, United Kingdom.

Pancreatic Polypeptide (PP) is a 36 amino acid peptide, secreted from the endocrine pancreas. It belongs to the PP fold family of peptides which also includes Neuropeptide Y (NPY) and Peptide YY (PYY). PP fold peptides act via Y receptors, a group of G-protein coupled receptors linked to adenylate cyclase. PP has the highest affinity for Y4 receptors, which show limited affinity for other PP fold peptides. Previous work has shown that administration of PP inhibits food intake in human volunteers and in rodent models. However, the short circulating half-life of PP limits its use as an anti-obesity agent. Determining the mechanisms by which PP is broken down by endogenous peptidases will allow the rational design of peptidase resistant analogues with greater clinical utility.

We investigated whether the peptidases dipeptidyl peptidase IV (DPPIV) and neprilysin (NEP) breakdown PP in vitro using high performance liquid chromatography and matrix assisted laser desorption/ionisation-time of flight mass spectrometry. In vivo, we examined the effect of co-administration of NEP or DPPIV inhibitors with PP on food intake and plasma levels of PP in mice. Finally, we determined whether PP analogues with amino acid substitutions at the positions our experiments suggested were targeted by DPPIV and NEP were resistant to breakdown by these enzymes in vitro and in vivo.Our results demonstrate that both DPPIV and NEP break down PP in vitro. Breakdown analysis of PP using MALDI-TOF MS suggests cleavage points at position 20 and 30 when incubated with NEP in vitro. The inhibition of NEP in vivo showed increased food intake inhibition after two hours following a dose of 150nmol/kg of PP. Plasma levels were increased in mice which received the NEP inhibitor, from 15,000fmol/ml PP alone to 35,000fmol/ml with NEP inhibitor at 45 minutes. Analogues with targeted substitutions to prevent break down by NEP and DPPIV have improved food intake inhibition in mice.

Degradation resistant PP analogues may have clinical utility in the treatment of obesity. The precise physiological mechanisms by which PP is broken down require further investigation.