Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression1

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Abstract

The regulation of glucose metabolism by glucagon and GLP-1 is well established, but novel functions for these and other proglucagon-derived peptides are less well defined. This paper highlights the diversity of both GLP-1 and glucagon activity by studying the tissue distribution of glucagon and GLP-1 receptor gene expression by both Southern blot analysis of RT-PCR products and nuclease protection assays. By Southern blot analysis of RT-PCR products, GLP-1 receptor mRNA was detected in lung, hypothalamus, hippocampus, cerebral cortex, kidney, pancreas, and throughout the gastrointestinal tract. Glucagon receptor expression was detected in liver, kidney, spleen, thymus, adrenal glands, pancreas, cerebral cortex, lung, and throughout the gastrointestinal tract. Nuclease protection assay revealed glucagon receptor expression to be highest in liver and kidney, whereas GLP-1 receptor expression was only detected by protection assay in lung, stomach, and large bowel. Despite previous evidence that other receptors for proglucagon-derived peptides may exist, no evidence of novel receptors or multiple isoforms of the glucagon and GLP-1 receptors was found, indicating that the two cloned receptors may mediate all the effects of proglucagon-derived peptides, or that novel receptors may share less homology with the glucagon and GLP-1 receptors than previously anticipated.

Introduction

In addition to glucagon, proglucagon contains the coding sequences for several peptides. Mammalian proglucagon is differentially post-translationally processed in the pancreas and intestine, such that the extended forms of glucagon (glicentin and oxyntomodulin), and the glucagon like peptides GLP-1 and -2 are produced in the intestine, while glucagon is the predominant product in the pancreas (Holst and Orskov, 1994).

Despite these multiple products, receptors for only two of the proglucagon-derived peptides have been cloned; one for glucagon (Jelinek et al., 1993) and one for GLP-1 (Thorens, 1992). Both these receptors belong to a subfamily of G-protein coupled receptors which includes the vasoactive intestinal peptide, calcitonin and PACAP receptors. It has been suggested that multiple isoforms of the glucagon and GLP-1 receptors may be produced. Maget et al. (1994)have cloned splice variants of the 5′ end of the glucagon receptor gene by RT-PCR, and suggest that at least two of these variants are translatable and relatively abundant in the liver. Additional splice variants occurring further downstream were not found, however the glucagon receptor gene has eleven introns and it remains possible that other splice variants are produced. Unlike the glucagon receptor the genomic organisation of the GLP-1 receptor remains to be elucidated and as yet no splice variants have been cloned.

Iwanij (1995)also argues that there are novel isoforms of the canine glucagon receptor. UV irradiation was used to cross-link radiolabeled glucagon to its receptors in canine liver and kidney. The size of these receptors were judged by electrophoretic mobility, and the renal glucagon receptors were found to be consistently larger than the hepatic receptors. Treatments with neuraminidase, endoglycosidase F, or N-glycanase failed to convert the renal receptor to the size of the liver receptor. Additionally proteolytic mapping of the two receptors revealed that the major domains of both proteins are similar. Hence it was suggested that the canine renal receptor represents a structurally unique isoform of the hepatic glucagon receptor (Iwanij, 1995).

Although the role of glucagon and GLP-1 in glucose metabolism is well established (Philippe, 1991, Habener, 1993) other roles for these peptides have only recently been postulated. In particular, GLP-1 has now been implicated in the clearance of macromolecules from lung airways (Richter et al., 1993) and the regulation of satiety (Turton et al., 1996). The additional roles being postulated for glucagon and GLP-1 indicate that the distribution of tissue expression of the glucagon and GLP-1 receptors may be wider than originally anticipated.

Proglucagon-derived peptides have also been implicated in bowel growth, and for many years it has been postulated that at least one of these peptides has trophic activity in the bowel (Bristol and Williamson, 1988, Holst, 1983). Several studies have suggested that proglucagon-derived peptides may play a role in intestinal adaptation (Bristol and Williamson, 1988, Bloom and Polak, 1983). Intestinal adaptation is the response of residual bowel to the loss of functional bowel via disease or surgery. The changes which occur during adaptation include intestinal dilatation, muscle wall hypertrophy, and mucosal hyperplasia (Riecken et al., 1989, Albert et al., 1990, Taylor et al., 1990). Massive small bowel resection (MSBR) is an animal model of intestinal adaptation where 70–80% of the small bowel is resected. It has been shown that proglucagon mRNA levels increase post-MSBR (Taylor et al., 1990, Rountree et al., 1992, Fuller et al., 1993). Recently it was demonstrated that GLP-2 has trophic activity in mouse intestine (Drucker et al., 1996), and it appears likely that this will be the proglucagon-derived peptide involved in intestinal adaptation.

In the current studies Southern analysis of RT-PCR products and S1 nuclease protection assays have been utilised to address the possibilities that: (1) novel but related receptors exist for proglucagon-derived peptides; (2) multiple isoforms of the cloned glucagon and/or GLP-1 receptors are expressed; (3) the two known receptors may be expressed more widely than initially suggested and; (4) the glucagon and/or GLP-1 receptor may be involved in the process of intestinal adaptation.

Section snippets

Isolation of total RNA

Total RNA was extracted from adult Sprague–Dawely rat tissues by the guanidine isothiocyanate/caesium chloride gradient method (Chirgwin et al., 1979). Each RNA preparation was made from an individual organ except for the adrenals where one RNA sample was prepared from pools of four to eight adrenals.

Massive Small Bowel Resection (MSBR)

Approximately 80% of the small bowel was resected and the remaining bowel re-anastomosed as described previously (Taylor et al., 1990). The rats were sacrificed 2, 4 or 7 days post-MSBR and total

RT-PCR

Using a combination of RT-PCR primers designed from the conserved regions between the glucagon and GLP-1 receptors (Table 1), glucagon receptor clones were isolated from corpus, terminal ileum, hypothalamus and liver. GLP-1 receptor clones were isolated from corpus and terminal ileum. A nucleotide substitution (G969 to A969) was consistently found in the GLP-1 receptor sequences compared to the published sequence (Thorens, 1992). This change altered the encoded amino acid Val323 to Ile323 which

Discussion

The very sensitive RT-PCR Southern blot analysis allowed the detection of glucagon and GLP-1 receptor gene expression in a wide range of rat tissues, highlighting the possible diversity of action of these two receptors. The wide tissue distribution of glucagon receptor gene expression was further validated with a nuclease protection assay. The nuclease protection assay allowed the quantitation of expression, and therefore more accurately reflects the levels of expression. Levels of receptor

Acknowledgements

The authors gratefully acknowledge the help of Dr Monique Wolvekamp with the massive small bowel resections, and thank Sue Panckridge for her contribution to the figures.

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    This work was supported by a grant from the Crohn's and Colitis Foundation of America.

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