Similarly both neofunctionalization and subfunctionalization

Similarly, both neofunctionalization and subfunctionalization likely occurred for the duplicated proglucagon genes. The change in function of GLP-1 would be a neofunctionalization, however in this case it acquired a redundant function – a function that largely overlaps with glucagon effects in the liver (Mommsen et al., 1987, Mommsen and Moon, 1990, Plisetskaya and Mommsen, 1996). However, subfunctionalization of GLP-1 function is correlated to the new function acquired by Gcgrb through neofunctionalization. Loss of the GLP-2 exon by gcgb contributes to the subfunctionalization of gcga for GLP-2 production, while retention of a conserved glucagon b and GLP-1b sequences by gcgb suggests complementary subfunctialization. The complex interplay between the two glucagons with Gcgra and the four hormones (two glucagons and two GLP-1s) with Gcgrb, which likely have slightly differing spatial and temporal expression and signaling patterns, potentially allows very fine tuning of glucose metabolism in fish. In 98 0 to the glucagon receptors, only a single glp2r gene was retained in teleost fish after the fish-specific genome duplication (Fig. 1 and Supplementary Table S1). A duplicated copy of glp2r was lost soon after the fish-specific genome duplications as the phylogeny generated from the single copy glp2r sequences is in accord with the expected relationships of these species. Similarly, the GLP-2 encoding exon was lost from the gcgb gene soon after the duplication of the proglucagon genes as all teleost fish gcgb genes lack this exon. These observations suggest that the loss of the GLP-2 exon by gcgb resulted in only a single GLP-2 peptide being produced, thus yielding redundant GLP-2 receptors, allowing the loss of one of the two glp2r genes. There is no evidence for a change in function (neofunctionaization) for either GLP-2 or Glp2r, although, these functions are still unknown. Characterization of proglucagon genes in cartilaginous fish yielded a few surprises. First, a novel glucagon-like peptide was identified that we named GLP-3. Our analyses suggest that GLP-3 originated in the early vertebrate, before the origin of cartilaginous fish and therefore was lost from the genome of the ancestor of tetrapods and ray-finned fish. This peptide was previously identified from pancreatic extracts of several species of cartilaginous fish (Conlon et al., 1989, Conlon et al., 1994), thus likely has a biological function. Intriguingly, a gene encoding a receptor similar to the glucagon receptors was also found in the genomes of cartilaginous fish that did not group with any other characterized receptor (Fig. 5) nor reside in a genomic neighborhood similar to any other receptor (Supplementary Fig. S4), raising the possibility that this gene encodes Glp3r. Secondly, characterization of cartilaginous fish proglucagon sequences suggest that 37 amino acid long GLP-1 peptides arose much earlier than previously appreciated. Mammalian GLP-1 is produced as a 37-amino acid-long peptide that is subsequently processed to remove 6 N-terminal residues to produce an active hormone (Mojsov et al., 1986, Mojsov et al., 1987). GLP-1 from ray-finned fish does not have this N-terminal extension (Irwin, 2001). Proglucagon from amphibians do have the N-terminal extension, which suggested that it was acquired on the tetrapod lineage after divergence from fish (Irwin et al., 1996, Irwin, 2001). As cartilaginous fish also have this N-terminal extension, this indicates that the N-terminal extension was acquitted very early in vertebrate evolution, and potentially is involved in the diversification of the function of the proglucagon-derived peptides. The loss of the N-terminal extension in the lineage leading to teleost fish was accompanied by a gain of the proglucagon gcga and gcgb genes with differing coding potential. The release of GLP-1a and GLP-1b, from Gcga and Gcgb, respectively, with different or similar biological potencies represents a novel mechanism to diversify the function of GLP-1 in regulating glucose metabolism in fish.