The in vivo biological activity of the
The in vivo biological activity of the four novel analogues was then systematically assessed in three animal models including normal control, high fat fed and ob/ob mice. As expected from earlier in vitro studies, all four glucagon receptor antagonists had minor effects on glucose or insulin concentrations when administered alone in each model. This is encouraging and highlights the safety and tolerability of such peptide-based glucagon antagonists. Moreover, all analogues displayed substantial abilities to suppress glucagon action in the animal models. However, there was some disparity in their efficacy in each model. Thus, desHis1Pro4-glucagon effectively annulled glucagon action in normal mice, but was less effective in high fat fed and genetic models of obesity-diabetes. However, a further substitution of Asp9 for Glu9, known to be an important residue for signal transduction pathways following receptor binding (Unson et al., 1987, Unson et al., 1989), generated an analogue with particularly prominent effects in both models of obesity-diabetes. The acylated derivatives of desHis1Pro4Glu9-glucagon were marginally less effective than the non-acylated parent analogue. This is unsurprising since fatty BIO-acetoxime derivatisation is known to encourage binding to circulating plasma proteins, which extends circulating half-life but reduces concentrations of free biologically active peptide (Green and Flatt, 2007). Interestingly, there was no clear difference between the effects of mid-chain and C-terminal acylation, implying that neither modification unduly affects the alpha-helical structure of glucagon, which is known to be important for receptor binding (Ahn et al., 2001). In both ob/ob obese and high fat fed mice it would not be surprising that elevated plasma glucagon would arise in these models of obesity-diabetes (Flatt et al., 1982, Gustavsson et al., 2011). Thus, it is possible that the variability of responses found using different analogues may in part be due to the combined effects of background basal glucagon along with exogenous glucagon administered. The major rationale for acylation of regulatory peptides is to extend the circulating half-life of the molecule (Lee et al., 2012). As such, a possible lower intrinsic in vivo biological activity should be compensated for by an increase in the circulating half-life of the acylated molecule. This was clearly evident from the pharmacodynamic studies carried out in normal mice in the current study. Thus, desHis1Pro4-glucagon still possessed glucagon receptor antagonistic actions for up to 2h after initial administration, and further substitution of Asp9 for Glu9 extended this to 4h. Moreover, both fatty acid dervatised molecules retained glucagon receptor antagonistic properties up to 8h after injection, with desHis1Pro4Glu9Lys12FA-glucagon still effective some 24h later. This is particularly remarkable, but is consistent with other studies adopting similar approaches with chemically modified related regulatory peptides (Irwin et al., 2010). Clearly, more detailed longer-term studies are required to fully delineate the efficacy, safety and therapeutic applicability of these peptide-based glucagon antagonists, which falls outside the remit of the current study, but initial observations are extremely encouraging. Notably, glucagon receptor knockout mice appear to have significantly increased circulating levels of the incretin hormone, glucagon-like peptide-1 (Gelling et al., 2003), which is currently used in the diabetic clinic (Vilsboll et al., 2009). Thus, glucagon receptor inhibition may have numerous beneficial therapeutic actions in patients with type 2 diabetes. In conclusion, the present study has demonstrated that desHis1Pro4Glu9-glucagon is a stable, potent and effective glucagon receptor antagonist. Further modification to engineer the acylated analogues desHis1Pro4Glu9Lys12FA-glucagon and desHis1Pro4Glu9Lys30FA-glucagon produces longer-acting molecules with more prominent beneficial biological effects. Consequently, fatty acid derivatised analogues of desHis1Pro4Glu9-glucagon have exciting therapeutic potential and there is encouraging evidence to support further longer-term pre-clinical studies that will progress such glucagon antagonists towards diabetes therapy.