br Materials and methods br

Materials and methods
Results
Discussion The global pandemic of T2DM has mandated a search for new types of therapeutic agent and several long-acting analogs of naturally occurring incretins, particularly derivatives of GLP-1, have already been adopted in clinical practice [29]. This study has provided evidence that zfGIP acts as an in vitro insulinotropic peptide when incubated with isolated mouse islets and with both rodent- and human-derived clonal β-cells (Fig. 1, Fig. 2, Fig. 3). The peptide also exhibits in vivo glucose-lowering and insulin-releasing actions when administered to mice together with a glucose load (Fig. 7). However, the potency and efficacy of zfGIP are appreciably less than those of human GIP and human GLP-1 so that the peptide does not show great potential for development into an agent for treatment of patients with T2DM. Treatment of high fat fed mice with a peptidase-resistant GIP-oxyntomodulin hybrid peptide was shown to lead to beneficial actions on glucose homeostasis and reduction in weight [30]. However, administration of 50nmol/kg body weight of zfGIP to overnight fasted mice did not induce a significant change in cumulative food intake over a 3h period (G. Graham, unpublished data) so that the peptide is unlikely to find application in promoting weight loss in obese patients with T2DM. Synthetic dual- or triple-agonist peptides combining combinations of GLP-1, glucagon and GIP activities have been studied extensively as potential agents for T2DM therapy [31], [32], [33], [34]. The use of selective receptor antagonists has indicated that in vitro insulinotropic activity of zfGIP on BRIN-BD11 T7 High Yield Cy5 RNA receptor may involve activation of both the GIP and GLP-1 receptors expressed in this cell line. Insulin release elicited by the peptide was slightly but significantly decreased after incubating BRIN-BD11 cells with the GIP receptor antagonist, GIP(6-30)Cex-K40[Pal] and the GLP-1 receptor antagonist, exendin-4(9-39) suggesting that zfGIP acts as a dual agonist at both receptors. The action of the peptide was not antagonised by the glucagon receptor antagonist [desHis1,Pro4,Glu9]glucagon amide suggesting that zfGIP does not target this receptor. Consistent with this conclusion, zfGIP stimulated a significant increase in cAMP production in CHL cells transfected with the human GLP-1 receptor but not in HEK293 cells transfected with the human glucagon receptor. CRISPR/Cas9-engineered INS-1 cells with GLP-1R and GIPR knock-outs have proved to be valuable in characterizing polyagonist peptides with potential for treating patients with T2DM [28]. Consistent with the receptor antagonist studies, both the GLP-1R KO and the GIP KO cells responded to incubation with zfGIP with an increase in the rate of insulin release whereas the corresponding human peptides were inactive. This result shows that zfGIP is acting as a dual agonist at the GLP-1R and GIPR in INS-1 cells as well as in BRIN-BD11 cells. Our data are consistent with an earlier report by Musson et al. [13] that zfGIP is able to activate the rat GIP receptor but do not support their conclusion that the peptide is not able to activate the rat GLP-1 receptor. The reason for this discrepancy is difficult to determine as the origin, purity and structure of the zfGIP used in the study of Musson et al. were not reported. A comparison of the primary structure of GIP, GLP-1, and glucagon in zebrafish and human (Fig. 8A) demonstrates that evolutionary pressure to conserve the amino acid sequence has been appreciably less with GIP than with the other two hormones. Somewhat surprisingly in view of its proposed interaction with the human GLP-1 receptor, zfGIP shows greater structural similarity to human glucagon (11 amino acid residues) than to human GLP-1 (10 amino acid residues) (Fig. 8B). However, the general mechanism of activation of class B G-protein coupled receptors involves binding of the C-terminal domain of the peptide ligand to the extracellular N-terminal domain of the receptor. This promotes interaction of the N-terminal region of the ligand with the juxtamembrane domain of the receptor, thereby stimulating intracellular signalling [35], [36]. It is significant, therefore, that zfGIP shares with human/rat/mouse GLP-1 the residues Ala2, Glu3 in the N-terminal domain and Lys20 and Arg30 in the C-terminal domain that are not found in the corresponding glucagon molecule.