Abstract

RationaleInsulin‐like peptide 5 (INSL5) is a hormone produced by enteroendocrine L‐cells in the colon that has recently been implicated in the control of metabolic homeostasis. However, research into its physiology has been hindered by the reported unreliability of commercially available immunoassays and additional detection assays would benefit this emerging field.MethodsPeptides from purified murine L‐cells and homogenates from both human and mouse colonic tissues were extracted by precipitating larger proteins with acetonitrile. Untargeted liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses, followed by database searching, were used to detect and identify various INSL5 gene derived peptides and characterise their precise sequence. A similar approach was developed to quantify INSL5 levels in primary intestinal culture supernatants after purification and concentration by solid‐phase extraction.ResultsMass spectral analysis of purified enteroendocrine cells and tissue homogenates identified the exact sequence of A and B chains of INSL5 endogenously expressed in L‐cells. Differences in the endogenously processed peptide and the Swissprot database entry were observed for murine INSL5, whereas the human sequence matched previous predictions from heterologous expression experiments. INSL5 was detected in the supernatant of human and mouse primary colonic cultures and concentrations increased after treatment with a known L‐cell stimulus.ConclusionsThe first LC/MS/MS‐based method capable of the detection and semi‐quantitative analysis of endogenous INSL5 using MS‐based techniques has been demonstrated. The methodology will enable the identification of stimulants for INSL5 secretion from murine and human primary colonic epithelial cultures.

Highlights

  • Insulin‐like peptide 5 (INSL5) is a member of the insulin/relaxin gene family sharing a common structural signature comprised of an A and B chain with a conserved cysteine bridging pattern (Figure 1).[1]

  • We considered the true A and B chains to be the matched peptides that were present at the highest levels, and shorter peptides were considered degradation products

  • This study describes the adaptation of a well‐characterised extraction method for enriching peptides from complex matrices to the lysis of sorted enteroendocrine cells and whole tissue extracts to analyse INSL5 content

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Summary

Introduction

Insulin‐like peptide 5 (INSL5) is a member of the insulin/relaxin gene family sharing a common structural signature comprised of an A and B chain with a conserved cysteine bridging pattern (Figure 1).[1] INSL5 is expressed at highest levels in the colon of both mice and humans, with lower expression found for example in thymus and testis[1,2] and. Within the colon it is expressed in enteroendocrine L‐cells,[3] which secrete glucagon‐like peptide‐1 (GLP‐1)[4] and peptideYY (PYY).[4] Liu and collaborators showed that INSL5 stimulates the G‐protein coupled receptor RXFP4 (GPR100 or GPCR142) but no precise physiological function of the peptide was initially identified.[5] Since INSL5 has been implicated in the regulation of metabolism, either by affecting glucose tolerance through an impact on insulin production,[6] insulin secretion[7] or the regulation of hepatic glucose production[8] or through

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