Structure, measurement, and secretion of human glucagon-like peptide-2

doi:10.1016/S0196-9781(99)00176-X

Peptides

Volume 21, Issue 1, January 2000, Pages 73-80

https://doi.org/10.1016/S0196-9781(99)00176-X Get rights and content

Abstract

By using radioimmunoassays toward the cDNA-predicted amino acid sequence of human glucagon-like peptide-2, a peptide was isolated from extracts of human ileum. By mass spectrometry and Edman sequencing, this peptide was identified as human proglucagon 126-158. High-performance liquid chromatography analyses indicated that a similar immunoreactive peptide (iGLP-2) was present in human plasma. Human plasma concentrations of iGLP-2 were elevated 3- to 4-fold at 1 to 2 h after ingestion of 800 to 1200 kcal meals.

Introduction

In mammals, the glucagon gene is expressed, not only in the pancreas, but also in the mucosa of the gastrointestinal tract [21], [24]. The single glucagon gene [1] encodes a common precursor, proglucagon (PG), a peptide of 160 amino acids that undergoes tissue-specific post-translational processing [10] (Fig. 1). Thus, in the pancreas, PG is predominantly cleaved to produce glicentin-related pancreatic polypeptide (GRPP), corresponding to PG 1-30, glucagon itself (PG 33-61) [22], [26], a hexapeptide corresponding to PG 64-69 (intervening peptide 1), and the so-called major PG fragment (MPGF; PG 72-158) [11], [28]. The processing pattern in the small intestine differs markedly. Here, the major secreted products are the 69 amino acid glucagon-containing peptide, glicentin, (PG 1-69) [29], the two glucagon-like peptides GLP-1 (PG 78-107NH₂) [12], [29] and GLP-2 (PG 126-158) [5], [30], and the so-called intervening peptide 2 (PG 111-123) [5].

So far, very little is known about GLP-2 [13]. However, Drucker et al. have recently shown that GLP-2 induces intestinal proliferation in mice [8], [27]. To investigate human GLP-2 in greater detail, we determined its exact chemical structure, developed a specific radioimmunoassay for fully processed GLP-2, and studied its secretion over a 24-h period in which healthy subjects received three mixed meals.

Section snippets

Peptides

Synthetic GLP-2 1-11 (PG 126-136), bovine GLP-2 with Thr¹²→Tyr¹² substitution, and recombinant human GLP-2 (PG 126-158) made in yeast, were all produced at Novo Nordisk (Bagsvaerd, Denmark). The structure and purity of these peptides were confirmed by high-performance liquid chromotography (HPLC), mass and sequence analysis.

Tissue extraction

Fresh pieces of human ileum and pancreas were obtained from transplantation donors (approved by the Local Ethical Committee) and immediately frozen.

The tissues were

N-terminal radioimmunoassay

The selected antiserum (code no. 92160) could be used in a final dilution of 1:35.000 and had a binding affinity for PG 126-159 of approximately 10¹⁰ l/mol (Scatchard analysis). It showed no cross-reaction with GLP-1 1-36NH₂, GLP-1 7-36NH_2, glicentin, oxyntomodulin, glucagon, pituitary adenylyl cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), growth hormone releasing factor (GHRF), gastric inhibitory peptide (GIP), secretin, and peptide histidine isoleucine amide

Discussion

The present study provides information about the exact chemical structure of human GLP-2 and its secretion as revealed by the peripheral plasma concentrations in healthy human subjects. Earlier studies on proglucagon processing in human intestine and pancreas revealed a differential processing of the precursor [32]. Thus, the pancreas was shown to contain and secrete mainly the so-called MPGF [28], [33], corresponding to residues no. 72 to 158 of proglucagon [11], in which the GLP-2 sequence is

Acknowledgements

We gratefully acknowledge the technical assistance of Allan Kastrup, Lene Albæk, and Rigmor Holck. This study was supported by The Danish Medical Research Council.

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¹: Present address: Dako A/S, Glostrup, DK-2600, Denmark.

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Regular PapersStructure, measurement, and secretion of human glucagon-like peptide-2

Abstract

Introduction

Section snippets

Peptides

Tissue extraction

N-terminal radioimmunoassay

Discussion

Acknowledgements

J Biol Chem

Gastroenterology

J Biol Chem

FEBS Lett

FEBS Lett

J Biol. Chem

Biochim Biophys Acta

J Biol Chem

FEBS Lett

Exon duplication and divergence in the human preproglucagon gene

Nature

Radioimmunoassay of gut regulatory peptides

Gut hormones in adaptation

Gut

Circulating and tissue forms of the intestinal growth factor, glucagon-like peptide-2

Endocrinology

Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo

J Clin Endocrinol Metab

Glucagon-like peptide 1 undergoes differential tissue-specific metabolism in the anesthetized pig

Am J Physiol

Induction of intestinal epithelial proliferation by glucagon-like peptide 2

Proc Natl Acad Sci USA

Regular Papers
Structure, measurement, and secretion of human glucagon-like peptide-2