evolution of receptors for peptides similar to glucagon

General and Comparative Endocrinology xxx (2014) xxx–xxx

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General and Comparative Endocrinology

journal homepage: www.elsevier .com/locate /ygcen

Evolution of receptors for peptides similar to glucagon

http://dx.doi.org/10.1016/j.ygcen.2014.03.0020016-6480/� 2014 Elsevier Inc. All rights reserved.

⇑ Address: Department of Laboratory Medicine and Pathobiology, University ofToronto, Room 6211 Medical Sciences Building, 1 King’s College Circle, Toronto, Ont.M5S 1A8, Canada.

E-mail address: [email protected]

Please cite this article in press as: Irwin, D.M. Evolution of receptors for peptides similar to glucagon. Gen. Comp. Endocrinol. (2014), http://dx.d10.1016/j.ygcen.2014.03.002

David M. Irwin ⇑Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont. M5S 1A8, CanadaBanting and Best Diabetes Centre, University of Toronto, Toronto, Ont. M5S 1A8, Canada

a r t i c l e i n f o

Article history:Available online xxxx

Keywords:GlucagonGlucagon-like peptidesGlucose-dependent insulinotropic peptide(GIP)ReceptorsEvolutionExendin

a b s t r a c t

The genes encoding the peptide precursors for glucagon (GCG), glucose-dependent insulinotropic peptide(GIP), and ortholog of exendin belong to the same family as shown by sequence similarity. The peptidessimilar to glucagon encoded by these genes signal through a closely related subfamily of G-proteincoupled receptors. A total of five types of genes for receptors for these peptides have been identified,three for the products of GCG (GCGR, GLP1R, and GLP2R) and one each for the products of GIP (GIPR)and the ortholog of exendin (Grlr). Phylogenetic and genomic neighborhood analyses clearly show thatthese genes originated very early in vertebrate evolution and all were present in the common ancestorof tetrapods and bony fish. Despite their ancient origins, some of these genes are dispensable, with theGlp1r, Gipr, and Grlr being lost on the lineages leading to bony fish, birds, and mammals, respectively.The loss of the genes for these receptors may have been driving forces in the evolution of new functionsfor these peptides similar to glucagon.

� 2014 Elsevier Inc. All rights reserved.

1. Proglucagon and secretin-like peptides

The proglucagon (GCG) gene encodes hormones that haveessential and differing roles in mammalian physiology. The threemajor hormones encoded by proglucagon are glucagon and thetwo glucagon-like peptides glucagon-like peptide-1 (GLP-1) andglucagon-like peptide-2 (GLP-2) (Kieffer and Habener, 1999;Drucker, 2005). Glucagon is the counter-regulatory hormone toinsulin and induces glucose production by the liver when bloodglucose levels are low (Jiang and Zhang, 2003; Ramnanan et al.,2011). GLP-1 is a major incretin hormone that potentiates insulinrelease by pancreatic islet beta cells in response to eating a meal(Meier and Nauck, 2005; Holst, 2007; Baggio and Drucker, 2007).GLP-2 has important roles in maintaining intestinal function(Drucker, 2001; Baggio and Drucker, 2007). The major actions ofthese hormones are non-overlapping and demonstrate the diverg-ing functions of related sequences (Kieffer and Habener, 1999;Drucker, 2005). In addition to these major physiological functions,these three glucagon-like hormones produced from proglucagonhave additional important activities, many of which are involvedin feeding behavior (Alvarez et al., 1996; Lovshin et al., 2004;Baggio and Drucker, 2007). The processing of proglucagon to

produce the glucagon-like hormones also yields additionalpeptides, e.g., intervening peptide-1 (IP1), which may have addi-tional physiological functions (Kieffer and Habener, 1999; Drucker,2005).

The proglucagon-derived hormones are members of a largerfamily of secretin-like hormones that are found to play diversephysiological roles in many metazoan species (Hoyle, 1998;Sherwood et al., 2000; Roch et al., 2009). Mammalian genomescontain six genes that encode a total of 10 secretin-like hormones(Hoyle, 1998; Sherwood et al., 2000; Roch et al., 2009). In additionto the proglucagon (GCG) gene, which encodes three secretin-likesequences (glucagon, GLP-1 and GLP-2), the adenylate cyclase acti-vating peptide (ADCYAP) and vasoactive intestinal peptide (VIP)genes each encode two secretin-like sequences. ADCYAP encodesthe hormones PACAP (pituitary adenylate cyclase activatingprotein) and PACAP-related peptide (PRP; sometimes calledgrowth hormone releasing hormone-like peptide, GHRH-LP) whileVIP encodes VIP and peptide histidine methionine (PHM) or pep-tide histidine isoleucine (PHI) (depending upon whether last aminoacid of the hormone in the species is methionine or isoleucine). Thethree remaining human genes each encode a single secretin-likesequence, and they are the genes for secretin (SCT), growthhormone releasing hormone (GHRH) and glucose-dependent insu-linotropic peptide (GIP). Additional secretin-like sequences havebeen identified in some vertebrate species including the exendins(Hoyle, 1998), which were first identified in the reptile Gila mon-ster and relatives (Heloderma suspectum and Heloderma horridum)

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2 D.M. Irwin / General and Comparative Endocrinology xxx (2014) xxx–xxx

(Raufman, 1996). Recently putative orthologs of the gene encodingexendin have been identified in other vertebrates, including otherreptiles, birds, amphibians and fish, but not in mammals (Irwin andPrentice, 2011; Irwin, 2012; Wang et al., 2012; Park et al., 2013).This gene has also been called glucagon-like (Gcgl) (Wang et al.,2012) and glucagon-related peptide (Gcrp) (Park et al., 2013).

Phylogenetic analysis of the secretin-like hormones yieldspoorly supported trees due to the short length of their peptide se-quences (Dores et al., 1996). However, phylogenetic analyses typi-cally show that the secretin-like peptides encoded by theproglucagon (glucagon, GLP-1 and GLP-2) and GIP genes are mostclosely related (Hoyle, 1998; Irwin et al., 1999; Cardoso et al.,2010; Ng et al., 2010; Irwin, 2012) (see Fig. S1). Exendin-1 and -2, from the Gila monster, are most similar to GLP-1, whileexendin-3 and -4 are very divergent and more similar to othersecretin-like hormones (Hoyle 1998; Sherwood et al., 2000; Fryet al., 2010). Comparison of the exendin-1 and -4 precursorsequences, although, showed that the non-hormone portion ofthe precursors (signal and N-terminal pro-peptide sequences) arevery similar (Pohl and Wank, 1998). These observations suggestedthat the exendin peptides experienced convergent evolution, withsome of the exendin sequences evolving to be more similar toeither GLP-1 or VIP (Fry et al., 2010). The inclusion of putativeorthologs of the exendin peptides (from other reptiles and birds)into phylogenetic analyses of secretin-like peptides allowed a bet-ter resolution of the relationships of exendin to the other secretin-like peptides revealing that exendin is most closely related to theproglucagon-derived peptides and GIP (Irwin and Prentice, 2011;Irwin, 2012; Wang et al., 2012; Park et al., 2013) (see Fig. S1). Thisrecent phylogenetic analysis of secretin-like peptides concludesthat exendin-1 and -2 evolved convergently upon the VIP sequence(Irwin, 2012). The close relationship of the exendin, GCG, and GIPgenes was strengthened by the observation that the genomic loca-tion of the exendin ortholog is similar to those for the GCG and GIPgenes (Irwin and Prentice, 2011; Irwin, 2012; Wang et al., 2012;Park et al., 2013). These observations suggest that GCG, GIP, andthe gene for the exendin ortholog represent three of the four genesgenerated during the pair of genome duplications that occurred onthe ancestral vertebrate lineage (Irwin and Prentice, 2011; Irwin,2012; Wang et al., 2012). The remaining genes encoding thesecretin-like sequences are not found in similar genomic neighbor-hoods, consistent with a conclusion that the GCG, GIP, andexendin ortholog genes are more closely related to each other thanthey are to any of the other secretin-like genes (Irwin and Prentice,2011).

2. Genes for the receptors for peptides similar to glucagon

Proglucagon-derived peptides and other secretin-like hormonesexert their physiological effects through binding to specific recep-tors found on the surfaces of cells in the target tissues. A cDNAclone for a specific receptor for the GLP-1 receptor (GLP1R) wascloned in 1992 and found to encode a G-protein coupled receptor(GPCR) (Thorens, 1992). Receptors for the other two glucagon-likepeptides encoded by GCG, glucagon (GCGR) and GLP-2 (GLP2R), arealso G-protein coupled receptors (Jelinek et al., 1993; Munroeet al., 1999), as are the receptors for the peptide similar to glucagonencoded by the GIP gene (GIPR; Usdin et al., 1993) and the putativeortholog of Gila monster exendin (GRLR, Irwin and Prentice, 2011;also called GCGLR, Wang et al., 2012; and GCRPR, Park et al., 2013).The genes for each of these receptors are expressed in a tissue-spe-cific manner, with high expression in the physiologically relevanttissues (i.e., tissues where the hormones have known or predictedphysiological action). While the function of the ortholog of exendinis currently unknown, the observation that the Grlr gene is mostabundantly expressed in the brain (Irwin and Prentice, 2011; Wang

Please cite this article in press as: Irwin, D.M. Evolution of receptors for pepti10.1016/j.ygcen.2014.03.002

et al., 2012) suggests that the likely physiological target of theexendin ortholog is in the brain, however, this still leaves a long listof potential functions. The discovery that GLP1R and GLP2R are alsoexpressed in portions of the brain (Alvarez et al., 1996; Lovshinet al., 2004) prompted searches for additional functions for thesepeptides. Receptors for the peptides similar to glucagon range insize from about 400 to 550 amino acids in length, with GIPR beingshortest and GLP2R being longest (see Figs. 1 and S2). Alignment ofthe protein sequences of these receptors show that the sequencesshow similarity across most of their length, with the regionsencoding the seven transmembrane domain regions being well-conserved, as are the distances between these seven domains,whereas the N-terminal and C-terminal regions show greater var-iability both in length and in their sequences (see Figs. 1 and S2).

The genes for the receptors for peptides similar to glucagon aredispersed on three human chromosomes, with two on chromo-some 17 (GCGR and GLP2R) and one each on chromosomes 6(GLP1R) and 19 (GIPR) (see Table S1). The exon–intron gene struc-tures of these genes are similar, as are the genes for other secretin-like hormone receptors, with the protein-coding region distributedover 13 coding exons (Fig. 1). The structure of the GCGR and GIPRgenes differ from those of the GLP1R and GLP2R by having an addi-tional upstream exon that only contains 50 untranslated sequence(Fig. 1). Introns between the coding exons are at very similar posi-tions in the alignment of the receptor protein sequences, with thehomologous introns interrupting the coding sequences in identicalphases (Figs. 1 and S2). With the exception of the first and last cod-ing exons, the lengths of the remaining coding exons are similaramong the receptor genes (Table S2).

In parallel with the identification of the orthologs of the Gilamonster exendin gene, an additional glucagon receptor-like gene,the glucagon receptor-like receptor (Grlr, also called Gcglr andGcrpr) was found in the genome sequence of many vertebrate spe-cies (Irwin and Prentice, 2011; Wang et al., 2012; Park et al., 2013).The initial study identifying this receptor gene (Irwin and Prentice,2011) hypothesized that the product of the exendin orthologshould be the ligand for this receptor, with subsequent studiesconfirming this interaction (Wang et al., 2012; Park et al., 2013).The human genome, like that of other mammals with availablegenome sequences, does not contain a GRLR gene, and to date thisgene has only been found in non-mammalian vertebrates (Irwinand Prentice, 2011; Wang et al., 2012; Park et al., 2013). The in-tron–exon structure of the Grlr gene, as well as the sizes of its cod-ing exons, is similar to those for the genes for other receptors forpeptides similar to glucagon (Fig. 1).

3. Receptors for peptides similar to glucagon and Class B1 of G-protein coupled receptors

Receptors for peptides similar to glucagon encoded by GCG, GIP,and the ortholog of the Gila monster exendin gene are GPCRs, asare the receptors for many other secretin-like peptides. Specificreceptors for secretin (SCTR), VIP (VPAC1 and VPAC2), GHRH(GHRHR) and PACAP (ADCYAP1R1) are all GPCRs (see Table S1)(Fredriksson et al., 2003; Fredriksson and Schiöth, 2005). The largeGPCR gene family consists of at least 850 genes in mammals(Fredriksson et al., 2003; Fredriksson and Schiöth, 2005; Bjarnadóttiret al., 2006; Krishnan et al., 2013). Phylogenetic analyses of GPCRgenes in a number of vertebrate species have consistentlyidentified a subset of GPCRs, designated the Class B1 GPCRs, whichinclude the receptors for the secretin-like peptides describedabove as well as receptors for corticotropin releasing hormone(CRHR1 and CRHR2), parathyroid (PTH1R and PTH2R), calcitonin(CALCR) and a calcitonin receptor-like gene (CALCRL) (Harmar,2001; Fredriksson et al., 2003; Fredriksson and Schiöth, 2005;

des similar to glucagon. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/



hGCGR (10kb, 477aa)

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hGLP1R (39kb, 463aa)

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hVIPR1 (35kb, 457aa)

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7 transmembrane domains

Fig. 1. Structure of the genes for receptors for peptides similar to glucagon. Schematic structure of the human GCGR, GLP1R, GLP2R, GIPR and VIPR1 and the chicken Grlr genes(see Table S1 for source of data). The VIPR1 gene is representative of the other class B1 GPCR genes. Exons are shown as boxes, with introns and flanking sequence as thin lines.The length of the gene, in kilobases, and number of amino acids in the encoded product are indicated. Filled boxes are coding region, while open boxes are untranslatedregions. Coding exons are proportional to size (see Table S2), while non-coding exons and introns are not. Numbers below each exon are the phase of the codons interruptedby the introns (see Sharp, 1981). The bar above the gene structures indicates the exons that encode the seven transmembrane domains.

D.M. Irwin / General and Comparative Endocrinology xxx (2014) xxx–xxx 3

Mayo et al., 2003; Glorian et al., 2007; Krishnan et al., 2013).Phylogenies of the nucleotide or amino acid sequences of thesereceptors consistently group the receptors for the secretin-likehormones as a monophyletic group with CRHR1, CRHR2, CALCR,and CALCRL being the most closely related sister groups (Cardosoet al., 2005, 2006; Ng et al., 2010; Tam et al., 2011; Park et al.,2013).

The phylogeny of the secretin hormone-like receptors typicallyseparate into two monophyletic groups: genes for the receptors forproglucagon-derived peptides (GCGR, GLP1R and GLP2R) and GIP(GIPR) forming one group; and genes for the receptors for the othersecretin-like hormones (SCTR, GHRHR, VIPR1, VIPR2, and ADC-YAP1R1) forming a second group (Cardoso et al., 2005, 2006; Nget al., 2010; Tam et al., 2011; Park et al., 2013). In Fig. 2 the phylo-genetic relationships of the human genes for receptors for secretin-like hormones, along with the chicken gene for the receptor for theexendin homolog, is illustrated (see Table S1 for source of data,Fig. S3 for the DNA sequence alignment, and Fig. S4 for the treegenerated by the maximum likelihood method in Newick format).Phylogenies generated by both Bayesian and maximum likelihoodmethods (see Figs. 2 and S4) confidently grouped the receptors forthe proglucagon-derived peptides, and GIP, and yielded strong sup-port for a second group that included all of the other secretin-likehormones. The chicken Grlr gene also confidently groups with thereceptors for the proglucagon-derived peptides and GIP, in agree-ment with previous studies (Irwin and Prentice, 2011; Park et al.,2013). The gene for the GIP receptor (GIPR) is most closely related(and thus have shared the most recent common ancestry) to theglucagon receptor gene (GCGR), with more ancient divergencesfrom this pair leading to Grlr, GLP1R, and GLP2R (Figs. 2 and S4).The phylogeny in Fig. 2 implies that the receptor genes divergedin a step-wise fashion with a gene for the GLP-2 receptor divergingfirst, followed by the divergence of genes for the GLP-1 receptor,then a gene for the receptor of the exendin ortholog, and finallythe divergence of the genes for the GIP and glucagon receptors.Earlier studies had concluded that the relationships among thegenes for the receptors did not mirror the phylogenetic relation-ships of the proglucagon-derived peptides and GIP (Irwin, 2005,2010), where GLP-1 and GLP-2 were most closely related, with glu-cagon and GIP diverging earlier (Irwin et al., 1999; Ng et al., 2010;Cardoso et al., 2010). However, it must be cautioned, as mentionedabove, that phylogenies generated for short peptides often cannot


be confidently resolved (Dores et al., 1996), and interestingly, a re-cent phylogenetic analysis (Irwin, 2012) that included diversesecretin-like hormones including GIP and exendin homologs, frommammals, birds and reptiles yielded a phylogeny (see Fig. S1) thatalmost mirrored the receptor phylogeny shown in Fig. 2.

4. Phylogenetic analysis of vertebrate receptors for peptidessimilar to glucagon

Genes for receptors for peptides similar to glucagon and othersecretin-like hormones have been identified in a number of verte-brate species (Sivarajah et al., 2001; Chow et al., 2004; Irwin andWong, 2005; Cardoso et al., 2005, 2006; Ng et al., 2010). Orthologyof genes was typically defined using the following approaches: se-quence similarity, phylogeny, or genomic neighborhood analysis,with comparison to the well-characterized receptor genes frommammalian species. The specific ligand-binding properties of onlya few of these receptors from non-mammalian species have beencharacterized (Ngan et al., 1999; Mojsov, 2000; Sivarajah et al.,2001; Yeung et al., 2002; Chow et al., 2004; Musson et al., 2009;Wang et al., 2012; Park et al., 2013).

Knowledge of which species possesses which receptors shouldallow a better understanding of the differences in the biology ofthese peptides similar to glucagon, and identify when these recep-tors may have originated. Here, to better understand the evolutionof this gene family we have expanded upon earlier studies bysearching all available genome sequences of vertebrate species inrelease 73 of the Ensembl database (www.ensembl.org), as wellas those in the PreEnsembl database (pre.ensembl.org) (searchesconducted in September 2013). Databases were searched using ap-proaches that we (Irwin and Wong, 2005; Irwin and Prentice,2011), and others (Cardoso et al., 2005, 2006; Ng et al., 2010), havepreviously used, with candidate genes identified in the Ensembldatabase listed in Table S3 and those from the Pre Ensembl data-base in Table S4. The searches resulted in the identification of alarge number of potential receptor genes. Due to the draft natureof many of the genome sequences in Ensembl, many of the genescould not be fully characterized, as they would either be missingpart of the gene or were distributed over several genomic contigs.This incomplete nature of these genomes reduces the abilityto clearly identify orthology as similarity is limited, genomic


http://www.ensembl.org

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Fig. 2. Phylogeny of class B1 G-protein-coupled receptors. Phylogeny of human Class B1 G-protein coupled receptors and the Chicken Grlr (see Table S1 for data source). Thetree shown here was generated using PhyML (Guindon et al., 2005) using sequences aligned with MAFFT (Katoh et al., 2002) and trimmed with Guidance (Penn et al., 2010).The Newick formatted tree file is in Fig. S4. The GTR plus gamma model was used for the maximum likelihood analysis and was selected as the best fitting evolutionary modelby ModelTest (Posada, 2006). The tree is a bootstrap consensus tree based on 100 replications. A similar tree, with identical topology, was identified using Bayesian methodsas implemented in MrBayes 3.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003; Ronquist et al., 2012). Numbers at the nodes are the maximum likelihoodbootstrap support followed by the Bayesian posterior probabilities. The CRHR1, CRHR2, CALCR and CALCRL genes were used as outgroups to root the tree. Receptors forpeptides similar to glucagon are indicated in bold, as is the support values for the monophyly of these receptor genes.


neighborhood analysis could not be used, and the partial sequencewere often not clearly resolved in phylogenetic trees (results notshown). Receptor genes listed as ‘‘not found’’ in Tables S3 and S4may not have been identified in a species solely due to thefragmented nature of the assembly rather than the gene trulybeing missing from the genome.

Sequence similarity is often used to identify related sequences,however, due to variation in rates of sequence evolution, the mostsimilar sequences are not necessary the most closely related(Stewart, 1993), since distantly related, but more slowly evolving,sequences can show greater sequence similarity than more closelyrelated, but more rapidly evolving, sequences. This phenomenoncomplicates the identification of receptor genes from differentclasses of vertebrates, and requires the use of methods that allowfor variation in the rate of sequence evolution. Several groups haveexamined the relationships for the genes for receptors for peptidessimilar to glucagon from diverse vertebrate species, sometimesresulting in differing conclusions (Sivarajah et al., 2001; Chowet al., 2004; Irwin and Wong, 2005; Cardoso et al., 2005, 2006;Ng et al., 2010; Irwin and Prentice, 2011; Park et al., 2013). Thesediffering conclusions can be attributed to using limited data, eitherin the number of sequences or incomplete sequences. The best res-olution of phylogenetic trees typically is achieved using the longestamount of sequence data, therefore we used only full-length, ornear full-length, sequences in the analysis presented here. Fig. S1shows an alignment of the protein sequences for the vertebratereceptors for peptides similar to glucagon used for our phyloge-netic analysis, while Fig. S5 is the alignment of the DNA sequencesin fasta format. While shorter sequences may result in trees withlower levels of support, and thus weaker phylogenetic conclusions,considerable success has been achieved when the well-conserved7-transmembrane core portion of the GCPRs (see Fig. 1) has beenused to construct phylogenies of these receptors (e.g., Sivarajahet al., 2001; Chow et al., 2004; Irwin and Wong, 2005; Cardosoet al., 2005, 2006). Indeed using the core transmembrane region


has allowed identification of potential orthologs in a representativeoutgroup to vertebrates the non-vertebrate Ciona intestinalis (tuni-cate) genome (Cardoso et al., 2006; Mirabeau and Joly, 2013).

Phylogenetic trees of receptors for peptides similar to glucagonfrom diverse vertebrate species typically are similar to the treepresented in Fig. 3 (Sivarajah et al., 2001; Chow et al., 2004; Irwinand Wong, 2005; Cardoso et al., 2005, 2006; Ng et al., 2010; Parket al., 2013; Hwang et al., 2013). The tree shown in Fig. 3 (seeFig. S6 for the tree in Newick format) was generated using the max-imum likelihood method with aligned coding sequences (seeFig. S5 for the DNA sequence alignment). Similar results were ob-tained with the Bayesian and neighbor joining methods (resultsnot shown), or if different outgroups or GLP2R was used as the out-group (see Fig. S7). Initial phylogenetic analyses (Sivarajah et al.,2001; Chow et al., 2004; Irwin and Wong, 2005; Cardoso et al.,2005, 2006; Ng et al., 2010) tended to only identify four types ofreceptors for peptides similar to glucagon, the GCGR, GLP1R, GLP2R,and GIPR genes, which corresponded to the known mammalianreceptors for peptides similar to glucagon. However, these analyseshad difficulty recovering the expected species phylogenies withinsome of the receptors classes (e.g., GIPR, Irwin and Wong, 2005;Ng et al., 2010), which was thought to be due to the use ofincomplete receptor gene sequences (Irwin and Prentice, 2011).In parallel with the discovery of orthologs of the Gila monsterexendin gene in diverse vertebrates, recent analyses haveidentified a fifth group of receptor genes, the Grlr genes (Irwinand Prentice, 2011; Park et al., 2013; Hwang et al., 2013). In retro-spect, it was found that genes were responsible for the inconsistentspecies phylogenies and inferred to be Gipr or Glp1r genes, wereactually Grlr genes. As shown in Figs. 3, S6, and S7 the monophylyof all five types of receptors for peptides similar to glucagon arewell supported, and yield topologies that are largely consistentwith expected species phylogenies. The initial identification ofthe Grlr receptors (Irwin and Prentice, 2011) led to the hypothesisthat it should be the receptor for peptides encoded by the exendin




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Fig. 3. Phylogeny of vertebrate receptors for glucagon-like peptides. Phylogeny was generated using PhyML version 3.0 (Guindon et al., 2005) using the coding sequences ofgenes for receptors for peptides similar to glucagon from diverse vertebrate species aligned with MAFFT (Katoh et al., 2002) and trimmed with Guidance (Penn et al., 2010).The Newick formatted tree file is in Fig. S6. Sequence data are from Tables S3, S4, and S5. The aligned DNA sequence generated from the alignment (prior to trimming) isshown in Fig. S5. The GTR plus gamma model was used for the maximum likelihood analysis, as it was the best fitting evolutionary model selected by ModelTest (Posada,2006). The bootstrapped tree was generated from 500 replications. Numbers at the nodes are proportion of bootstrap replicates supporting the node. A similar tree wasidentified using Bayesian methods as implemented in MrBayes version 3.2.2 or if additional outgroups were added (results not shown) or if GLP-2 sequences were used toroot the tree (Fig. S7). Groups of genes are indicted on the right, with the nodes that support these groups shown in bold. Glucagon receptors that likely also bind GLP-1 in fishare indicated as fish Glp1r. Softshell turtle refers to the Chinese softshell turtle.


gene orthologs, a hypothesis that was confirmed by subsequentstudies (Wang et al., 2012; Park et al., 2013).

Phylogenetic trees of the receptor genes show that GCGR andGLP2R are found in all vertebrate classes examined, however Glp1r,Gipr, and Grlr were not found in fish, birds, and mammals, respec-tively (Figs. 3, S6, and S7). The absence of a Glp1r in fish (Chowet al., 2004; Irwin and Wong, 2005) was a bit of a surprise asGLP-1 has a physiological function in fish (Plisetskaya and Momm-sen, 1996; Polakof et al., 2012), and cDNA clones that encodereceptors that can be activated by GLP-1 had been characterizedin zebrafish and goldfish (Mojsov, 2000; Yeung et al., 2002). Thefish receptors that are activated by GLP-1 are G-protein coupledreceptors from class B1, but are more closely related to GCGR thanto GLP1R (see Figs. 3, S6, and S7). Fish Glp1r genes are derived froma product of the duplication of the fish Gcgr gene, which likely wasdue to the fish specific genome duplication (Meyer and Van dePeer, 2005). Fish GLP-1 acts like a glucagon hormone rather thanan incretin (Plisetskaya and Mommsen, 1996; Polakof et al.,2012), thus the fact that the GLP-1 receptor in fish is duplicate of


the Gcgr explains the similarity of the physiological functions ofthese two hormones. The absence of Gipr from birds remains a puz-zle as the gene for the ligand (Gip) exits (Irwin and Zhang, 2006),however the physiological function of GIP in birds is not known.The gene for the ligand for Grlr has been lost in mammals, thusthe loss of the genes for both the ortholog of exendin and its recep-tor (Grlr) may provide an example of co-evolution between recep-tor and ligand.

5. Orthologous genes for receptors of peptides similar toglucagon reside in conserved genomic neighborhoods

To strengthen a conclusion of orthology, comparisons of thegenomic neighborhoods around genes can be conducted. If genesare orthologous, then one might expect that a larger genomic re-gion will be orthologous and that this sequence will containadditional genes that will also show a pattern of orthology. Thatis genes physically close to gene X in the genome of one species





should be orthologous (and similar) to genes near orthologs ofgene X in a different species. This approach has proved useful forfinding orthologous genes in divergent species, despite these geneshave limited sequence similarity due to increased rates of se-quence evolution. For this approach to be successful, gene ordernear the gene of interest must be conserved, and at least one ofthese genes needs to evolve a slow enough rate such that theorthologs in divergent species can be identified. This approachwas used to identify genes such as Lep (leptin) and Gip in fish,where the sequences for these genes could not easily be identifiedby genomic BLAST searches when mammalian gene sequenceswere used as queries (Kurokawa et al., 2005; Irwin and Zhang,2006). Strong evidence in support of the conclusion of gene losscan also be obtained from this approach. If the order of the flankinggenes were retained among several species, while the gene ofinterest cannot be found in one of the genomic sequences, then,this would suggest that the gene was deleted from the genomewhere the gene cannot be found. A good example of value of thisapproach was the detection of the specific loss of the Gckr (gluco-kinase regulatory protein) gene in birds (Wang et al., 2013).

Analysis of genomic neighborhoods has helped resolve ques-tions concerning the orthology of receptors for peptides similarto glucagon (Ng et al., 2010; Irwin and Prentice, 2011; Wanget al., 2012; Hwang et al., 2013). Using a diverse array of specieswith available genome sequence (see Fig. 4) the genomic neighbor-hoods surrounding the GCGR, GLP1R, GLP2R, GIPR and Grlr geneswere examined (Figs. 5–9). Similar genomic neighborhoods canbe seen for many of the receptor genes in the divergent species.For example, for GCGR, orthologous genes can be seen in most spe-cies shown in Fig. 5, however there are some notable differences.First, none of the genes near the presumptive lamprey Gcgr geneare orthologous to genes adjacent to GCGR in any other species.The lamprey Gcgr gene is on a shorter genomic contig with littlegene content and an incomplete sequence, limiting the ability to

Human

Lizard

Chicken/Duck

Turtle

Xenopus

Coelacanth

Gar

Zebrafish/Tilapia/Stickleback

Lamprey

Fig. 4. Phylogeny of species used for the genomic neighborhood analyses.Schematic relationship of species used in the genomic neighborhood analysesshown in Figs. 5–9. Branches indicate relationships and not lengths of time. Thedivergence of the two reptile groups (lizard and turtle) are shown as unresolvedfrom the divergence of the bird (duck and chicken) lineage. Zebrafish, tilapia, andstickleback are three different fish species used in the genomic neighborhoodanalyses and all are descendent of an ancestor that experienced a genomeduplication (indicated by the diamond). The gar lineage did not experience thegnome duplication.


accurately identify orthology. Based on sequence similarity andphylogenetic analysis (not shown), of the partial gene sequence,it was concluded to be a Gcgr ortholog, but the possibility remainsthat it is a paralogous gene duplicate, potentially one that was notretained in jawed vertebrates. In other species, such as the duckand lizard, despite sharing a large number of orthologous genes,some genes linked to Gcgr are missing or different. The Fam195bgene is missing from the duck chromosome, although whether thisis due to gap in the genome assembly or a true loss of the gene can-not be resolved. In the lizard, the gene, Ddx39a is linked to Gcgr,which may reflect a change in the organization of genes on thischromosome. Indeed more divergent species typically have greaterchanges in gene order and composition. In teleost fish, two Gcgr-like genes are often seen, and comparison of the genomic neigh-borhoods of these genes suggest that they were duplicated as partof a large genomic event, likely as a consequence of the fish-spe-cific genome duplication (Meyer and Van de Peer, 2005).

Intriguingly, in the coelacanth the Gcgr and Glp1r genes areadjacent to each other (Figs. 5 and 6). This was the only instancewhere a pair of receptor genes was found adjacent to each other.Glp1r genes had previously been identified in tetrapod vertebrates(mammals, reptiles, birds, and amphibians) but not in bony fish.Using genomic neighborhood analysis it is clear that the Glp1rgenes in tetrapods are orthologous, as similar sets of adjacentgenes are found (Fig. 6). The coelacanth Glp1r gene is incomplete,but based on sequence similarity and phylogenetic analysis (notshown) it appears to be most closely related to the Glp1r genes.Whether the unexpected genomic neighborhood of this gene isdue to a reorganization of the genome, incorrect assembly, orincorrect conclusions concerning its orthology requires additionalanalysis. No gene most closely related to GLP1R was found in thebony fish, either in representative species that diverged before(gar) or after (e.g., zebrafish) the fish-specific genome duplication(Tables S3 and S4). Searches of the fish genomes for orthologs ofgenes adjacent to the tetrapod Glp1r genes failed to find a con-served genomic neighborhood, as these genes were dispersedacross different chromosomes in the fish genomes. The failure tofind a conserved genomic neighborhood in fish may indicate eitherthis chromosomal region was subjected to large amounts of recom-bination in fish, or was assembled in a contiguous fashion only inthe ancestor of tetrapods. While a candidate Glp1r was identifiedin lamprey, this sequence was on a short genomic contig with noadjacent genes, is equally similar to the Grlr genes and yieldedambiguous phylogenetic placement, and thus, cannot be confi-dently classified.

The GLP2R gene appears to be in single copy gene throughoutvertebrates (Tables S2 and S3, Figs. 3 and 7, S6, and S7). Indeed,it is the only receptor gene in the lamprey that has an adjacentgene that is also adjacent in mammals and other vertebrates(Fig. 7), thus is the most confidently classified lamprey gene for areceptor of a peptide similar to glucagon. A conserved genomicneighborhood was identified in most species examined, althoughsome recombination has likely occurred rearranging gene orderfor some genes in some species (Fig. 7). Due to the fish-specificgenome duplication (Meyer and Van de Peer, 2005), one might ex-pect that there should be two Glp2r genes in teleost fish, however;only one Glp2r-like gene was found in the BLAST searches (TablesS3 and S4). As shown in Fig. 7, a pair of chromosomes were foundin zebrafish that are homologous to the single copy Glp2r bearingchromosome in the gar, a species that did not experience this gen-ome duplication. While one of the chromosomes contains a Glp2rgene, the other does not. A similar organization was found in otherfish genomes (data not shown), suggesting that the second fishglp2r gene was lost prior to the radiation of teleost fish.

GIPR genes were not found in any of the available bird genomeas well as the genomes of several species of fish (Tables S3 and S4).




GCGR FAM195B P4HBSLC25A10MRPL12HGS ARHGDIA

Human Chr 17

PPP1R27

Gcgr P4hbPpp1r27Slc25a10Hgs

Gar Chr LG12

Gcgr Glp1rMrpl12Hgs

Coelacanth Scaffold JH126596.1

Fam195b

Gcgr Fam195b P4hbSlc25a10Mrpl12Ddx39a Arhgdia

Lizard Chr 2

Ppp1r27

Gcgr Fam195b P4hbMrpl12Hgs

Xenopus Scaffold GL172918.1

Ppp1r27

Gcgr P4hbSlc25a10Mrpl12Hgs Arhgdia

Duck ScaffoldKB742551.1

Ppp1r27

Gcgr B4galnt

Lamprey Scaffold GL476704 ?

March4

Gcgr C3Slc25a10CldnRasd

Tilapia Scaffold GL831216.1

Tubb

Gcgr Trimm35 ASF1BMrpl12Hgs

Tilapia Scaffold GL831149.1

Tubb

Fam195b

170kb

325kb

60kb

240kb

250kb

300kb

210kb

170kb

250kb

Fig. 5. Genomic neighborhoods near the GCGR gene in diverse vertebrates. Organization of genes near GCGR in the human, duck, lizard, Xenopus, coelacanth, gar, tilapia, andlamprey genomes. Chromosome or genomic scaffold names are indicated after each species name. The length of the compared genomic fragments is indicated to the right.Gene names, as annotated from the human genome, are shown above the arrows that indicate direction of transcription. Gene sizes and distance between genes is not toscale. Orthologs of GCGR are labeled in blue. Other genes that show orthologous relationships distributed across vertebrates are labeled in red, while genes labeled in greenare those shared by the tilapia chromosomes. Two chromosome regions are represented for tilapia due to the fish specific genome duplication. The question mark (?) by thelamprey indicates that orthology is uncertain.

GLP1R SAYSD1 KCNK17DNAH8GLO1 KIF6

Human Chr 17

KCNK5

Glp1r Gcgr HgsFam195b


MrpL12

Glp1r Saysd1 Kcnk17Dnah8 Kif6

Lizard Chr 1

Kcnk5

Glp1r Saysd1 Ftsjd2


Kcnk5

Glp1r Saysd1 Kcnk17Dnah8 Kif6

Chicken Chr 3

Kcnk5

Glp1r/Grlr


Glo1

1Mb

600kb

700kb

200kb

350kb

30kb

Fig. 6. Genomic neighborhoods near the GLP1R gene in diverse vertebrates. Organization of genes near GLP1R in the human, chicken, lizard, Xenopus, coelacanth, and lampreygenomes. Chromosome or genomic scaffold name is indicated after each species. The length of the compared genomic fragments is indicated to the right. Gene names, asannotated in the human genome are shown above arrows that indicate direction of transcription. Gene sizes and distance between genes is not to scale. Orthologs of theGLP1R are labeled in blue. Other genes that show orthologous relationships distributed across vertebrates are labeled in red. A Glp1r gene was not found in the fish genomes.The lamprey gene is equally similar to both GLP1R and Grlr genes, thus is labeled with a question mark (?).


Please cite this article in press as: Irwin, D.M. Evolution of receptors for peptides similar to glucagon. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.002



GLP2R RCVRN MYH13RP11-477N12.3DHRSC7CUSP43

Human Chr 17

GAS7

Glp2r Pts Myh13Rp11-477n12.3Uts2rOgfod3

Gar Chr 17

Glp2r Pts


Glp2r Myh13Rp11-477n12.3Dhsc7cNarf

Lizard Chr 2

Gas7

Glp2r Myh16 Myh13Rp11-477n12.3Trim8Trim8


Trim8

Glp2r Myh13Rp11-477n12.3Dhsc7cZnf250

Chicken Chr 18

Gas7

Glp2rR Rcvrn

Lamprey Scaffold GL484465

Rcvrn Arl16Rp11-477n12.3Elval3

Zebrafish Chr 3

Gas7

Glp2r RcvrnRp11-477n12.3Hs3st3lDhrs7c

Zebrafish Chr 12

Pts

700kb

450kb

320kb

350kb

450kb

500kb

160kb

1Mb

30kb

Fig. 7. Genomic neighborhoods near the GLP2R gene in diverse vertebrates. Organization of genes near GLP2R in the human, chicken, lizard, Xenopus, coelacanth, gar,zebrafish, and lamprey genomes. Chromosome or genomic scaffold name is indicated after each species. Two chromosomal sequences are shown in zebrafish due to the fishspecific genome duplication, however a Glp2r gene was not found on one of the homologs. The length of the compared genomic fragments is indicated to the right. Genenames, as annotated in the human genome are shown above arrows that indicate direction of transcription. Gene sizes and distance between genes is not to scale. Orthologsof the GLP2R are labeled in blue. Other genes that show orthologous relationships distributed across vertebrates are labeled in red, while genes labeled in green are thoseshared among fish.


The genomic neighborhoods surrounding the GIPR genes in speciesthat have them was generally conserved (see Fig. 8), and likeGLP2R, those fish that did have a Gipr gene had only a single copyof the gene. As with GLP1R, genes adjacent to GIPR were not foundin a conserved organization in birds, thus why this gene is missingin these species is unclear. A potential Gipr gene exists in the lam-prey, but due to being on a short genomic contig its genomic neigh-borhood is unknown (Fig. 8) and its phylogenetic placement isweak (data not shown). The Grlr gene is missing in mammals,but exists in a largely conserved genomic neighborhood in speciesof many other vertebrate lineages (Fig. 9). In contrast to GLP1R andGIPR, a conserved genomic neighborhood can be found in mam-mals, however this region does not have a GRLR and instead hasan insertion of about 1 Mb of DNA. As mentioned above, a potentialGrlr gene may exist in the lamprey, but a more complete assemblyis required to confirm this.

6. Limitations in the analyses of genes for receptors of peptidessimilar to glucagon

Phylogenetic and genomic neighborhood analyses are powerfultechniques for identifying orthologous genes, as illustrated above.However, these approaches did not allow us to classify every genesequence that was identified in our BLAST searches into an orthol-ogy group (see Tables S3 and S4). A major limitation, of this, andmany other large-scale phylogenomic studies is the quality of thegenome assemblies used for the searches. Many genomes repre-sented in the Ensembl database are draft assemblies and of low


sequence coverage depth. The draft nature of many genomes re-sults in many genomic contigs containing gaps of undeterminedsequence. For a large number of our identified genes (Tables S3and S4) exons were not found as they likely reside in unsequencedgaps. Similarly, many genes were truncated, as an end of a genomiccontig would be in the middle of a gene. For some of these genes, asecond contig could be found that containing additional exons forthat gene, thus, a potential gene can be predicted, however, there isthe possibility that the two (or more) parts may actually representdifferent genes that would now be artificially put together. Due toincomplete nature of many genes, only a fraction of the total generepertoire of the receptors for peptides similar to glucagon encodesfull-length sequences that were used for our phylogenetic analysis.Many important species, including platypus, Xenopus tropicalis, andlamprey, who have only draft genome sequences, did not yield in-tact full-length genes that could be used for our analysis presentedin Figs. 3, S6 and S7.

Analysis of genomic neighborhoods of genes is even more lim-ited by the incompleteness of genome assemblies. Genes must re-side in a relatively large genomic contig so that it would containadditional genes for these analyses. As described above, many ofthe species had draft quality genome sequences, thus it was notpossible to identify large genomic contigs for many of the genesfor receptors for peptides similar to glucagon. The platypus andlamprey represent two important species, and there are many oth-ers, where the average contig size is short, and thus were not veryuseful for the genomic neighborhood analysis. Improvements inthe completeness and quality of genome assemblies are requiredto improve these types of analyses.




GIPR SNRPD2 FBXO46EML2GPR4OPA3

Human Chr 19

QPCTL

Gipr Cyp28 Alkbh6Eml2Gpr4Nova2

Gar Chr LG2

Clip3

Gipr Fbxo46 Pglyrp1Eml2Gpr4Opa3


Qpctl

Gipr Ccdc61Eml2

Turtle Scaffold JH210274.1

Pglyrp1

Gipr Fbxo46 Pglyrp1Eml2Gpr4Opa3 Snrpd2


Qpctl

Gipr


Gipr Clip3 Tmem87aEml2Gpr4Cd3eap Capns1b

Zebrafish Chr 17

Dharma

200kb

200kb

200kb

400kb

400kb

8kb

350kb

Fig. 8. Genomic neighborhoods near the GIPR gene in diverse vertebrates. Organization of genes near GIPR in the human, turtle, Xenopus, coelacanth, gar, zebrafish andlamprey genomes. Chromosome or genomic scaffold name is indicated after each species. The length of the compared genomic fragments is indicated to the right. Genenames, as annotated in the human genome are shown above arrows that indicate direction of transcription. Gene sizes and distance between genes is not to scale. A Gipr genewas not found on the homologous zebrafish chromosome generated by the fish-specific genome duplication. The Gipr gene was also not found in birds. Orthologs of the GIPRare labeled in blue. Other genes that show orthologous relationships distributed across vertebrates are labeled in red, while genes labeled in green are those shared by bonyfish. The question mark (?) by the lamprey indicates that orthology is uncertain.

1 Mb genomicDNA insertion

ANKS3 ZNF500CLUAP1NRLC3SLX4

Human Chr 16

C16orf71

Grlr H1f0Claup1Nrlc3

Gar Chr LG3

Rhot1

GrlrClaup1Nrlc3Abca3


Anks3

Grlr Anks3 Xpo6Cluap1Nrlc3Luc7l

Lizard Scaffold GL343518.1

C16orf71

Grlr Sbk1Claup1Nrlc3Slx4 Xpo6


Anks3

Grlr Anks3 Xpo6Cluap1Nrlc3Nmral

Chicken Chr 14

Sbk1

Grlr/Glp1r


Grlr RhotNrlc3Lcnt1Arhga H1f0

Stickleback Group V

Wdr90

1.1Mb

300kb

500kb

400kb

700kb

200kb

30kb

100kb

Fig. 9. Genomic neighborhoods near the Grlr gene in diverse vertebrates. Organization of genes near Grlr in the human, chicken, lizard, Xenopus, coelacanth, gar, stickleback,and lamprey genomes. Chromosome or genomic scaffold name is indicated after each species. The length of the compared genomic fragments is indicated to the right. Genenames, as annotated in the human genome are shown above arrows that indicate direction of transcription. Gene sizes and distance between genes is not to scale. Grlr geneswere not found in mammals (represented by human) or on one of the homologous stickleback chromosomes generated by the fish-specific genome duplication. Orthologs ofthe Grlr are labeled in blue. Other genes that show orthologous relationships distributed across vertebrates are labeled in red, while genes labeled in green are those shared bybony fish. The question mark (?) by the lamprey indicates that orthology is uncertain.


Please cite this article in press as: Irwin, D.M. Evolution of receptors for peptides similar to glucagon. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.002




In addition to the incomplete gene sequences the Ensembl data-base does not represent all major lineages of vertebrates, for exam-ple there is no representative of cartilaginous fish. While a genomesequence of a cartilaginous fish, the Elephant shark (Callorhinchusmilii), has been sequenced and published (Venkatesh et al., 2007)it is not completely assembled (nor is it available in the Ensembldatabase). We did search the Elephant shark genome, but onlyshort incomplete gene sequences that were not useful for ouranalyses were found (results not shown). Completion of additionalgenome sequences, especially from species that representvertebrate groups currently not represented in Ensembl, as wellas completion of existing draft genome sequences will likely yieldadditional surprises in the evolution of this and many other genefamilies.

7. Evolution of the genes for receptors of peptides similar toglucagon

The availability of complete genome sequence greatly enhancesour understanding of the evolution of genes and the physiologicalsystems generated by the products of these genes. Having com-plete genomes should allow identification of all related genes ina genome. Application of phylogenetic and genomic neighborhoodanalyses to genes from diverse vertebrate species has resulted ingreater understanding of the evolution and diversity of peptidessimilar to glucagon and their receptors. Searches of diverse verte-brate genomes have shown that both the diversity of genes encod-ing peptides similar to glucagon and receptors for these peptidesare larger than previously appreciated (Irwin and Prentice, 2011).These searches have also shown that the origin of both the diversepeptides similar to glucagon and their receptors were early in ver-tebrate evolution. While the GCG, GIP, and the ortholog of exendingenes likely originated via the genome duplications in an early ver-tebrate (Irwin et al., 1999; Irwin 2002, 2012; Hwang et al., 2013),the mechanism for the origin of the genes for the diverse receptorsfor peptides similar to glucagon is not as clear. Genome duplica-tions should yield duplicate genes that have neighboring genesthat are also related, similar to the conservation of genomic neigh-borhoods between species. While this is found for the GCG, GIP andortholog of exendin genes (Irwin et al., 1999; Irwin 2002, 2012;Hwang et al., 2013) none of the genes flanking any of the genesfor receptors for peptides similar to glucagon show any relation-ship. Whether this is because the genes originated via alternativemechanisms, or whether the conservation has been disrupted byrecombination is not known (Hwang et al., 2013). Despite notknowing the mechanism by which the receptor genes originated,it is clear from the phylogenetic (Figs. 3, S6, and S7) and genomicneighborhood analyses (Figs. 5–9) that they originated prior tothe divergence of bony fish and tetrapods, and likely before theemergence of jawless fish. Analyses of the genes for receptors forpeptides similar to glucagon from Ciona intestinalis (tunicate) indi-cate that the receptor family radiated after the tunicate-vertebratedivergence (Cardoso et al., 2006; Mirabeau and Joly, 2013).

After the origin of the family of genes for receptors for peptidessimilar to glucagon, the genes have not remained static. Not onlyhave their sequences evolved, but also like peptides similar to glu-cagon (Irwin, 2001, 2009, 2012; Ng et al., 2010; Hwang et al.,2013), the numbers of genes for the receptors has changed on dif-ferent vertebrate lineages (Irwin and Wong, 2005; Ng et al., 2010;Irwin and Prentice, 2011; Hwang et al., 2013). The Glp1r gene waslost on the lineage leading to teleost fish before the fish-specificgenome duplication, but then this genome duplication lead tothe generation of a redundant Gcgr genes, one of which gainedthe ability to be bound and activated by GLP-1, resulting in achange in the function of GLP-1 (Chow et al., 2004; Irwin andWong, 2005). Similarly, Gipr has been lost on the bird lineage,


while the Gip gene, encoding its ligand, has been retained (Irwinand Zhang, 2006), suggesting that GIP may interact with a differentreceptor in birds, and thus likely has a different physiological func-tion. In contrast, the Grlr gene was lost on the mammalian lineage,likely in parallel with the loss of the gene for its ligand. These genelosses should have resulted in the loss of the physiological functionof the ortholog of exendin.

Acknowledgments

Work from the author’s lab has been supported by Grants fromthe Natural Sciences and Engineering Research Council and theCanadian Institutes for Health Research.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ygcen.2014.03.002.

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evolution of receptors for peptides similar to glucagon

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