LAUSR

The discovery of glucagon-like peptide-1 (GLP-1) and demonstration of its insulin-releasing and glucose-lowering properties represent important milestones leading to the present drug exploitation of the GLP-1 receptor (GLP-1R) as a therapeutic target for both type 1 and particularly type 2 diabetes. This journey was initially thwarted by the short half-life of native GLP-1 (7-36) in the circulation, which necessitated continuous infusion of the peptide. It was not until the serendipitous finding that exendin-4 from the saliva of the Gila monster was both an agonist of the human GLP-1R and resistant to degradation by dipeptidyl peptidase-4 (DPP-4) that the therapeutic potential of GLP-1R activation could be clinically exploited. Thus, although exendin-4 shows only 53% sequence identity with human GLP-1, it was a full agonist at mammalian GLP-1R, with amino acid sequence at the N-terminal of His-Gly-Glu rather than His-AlaGlu. This conferred enzyme resistance to cleavage by DPP-4 that otherwise cuts between Ala-Glu, generating GLP-1 (9-36), which is inactive or even a mild GLP-1R antagonist. The demonstration of its glucose-lowering properties in humans was quickly followed by full exploitation of the GLP-1R as a target in type 2 diabetes using synthetic exendin-4 under the trade name of Byetta. Initially this was administered twice daily, but has since been replaced by modified forms facilitating only once-weekly administration. Other enzymeresistant forms of human GLP-1 followed onto the market, using acylation and/or amino substitution at position 9 in the N-terminus to confer DPP-4 resistance. The Gila monster has thus served people with diabetes well and kept the pharmaceutical industry busy for 30 years producing new generations of GLP-1R mimetics of different compositions, pharmacokinetic profiles and with multiple extrapancreatic actions useful for the treatment of diabetes and a range of other degenerative disorders. However, despite these developments, we have not fully appreciated the contribution of the Gila monster and addressed the obvious question of why such a metabolically active peptide is present in its saliva? Although it is not altogether unusual for regulatory peptides to be found at low concentrations in the saliva of humans, the large amount of exendin-4 in Gila monster saliva is unlikely to have evolved by chance and must have some functional purpose. Interestingly, this is not the creature's own form of GLP-1, which can be found in its intestinal L-cells with a structure very similar to mammalian GLP-1, including susceptibility to degradation by DPP-4. In this context, it is often cited that exendin-4 plays some role for the Gila monster in immobilization of its prey. Thus, when the lizard bites its prey, often small mammals, this introduces exendin-4 into the bloodstream which, as a result of insulinotropic action, induces hypoglycaemia and allows the prey to be overcome and devoured. This is, of course, highly unlikely for several reasons. First, a considerable dose would be required. Second, the effect of exendin-4 on insulin release, like that of GLP-1, is glucose-dependent and unlikely to cause hypoglycaemia. Third, even if this did occur, some considerable time would be required to incapacitate the prey, but this is in fact mediated very rapidly by toxins targeting the nervous and cardiovascular systems. In view of the above, we should consider an alternative possibility based on the fact that the Gila monster produces copious amounts of saliva when eating meals which can be up to one-third of its own body weight. The saliva containing exendin-4 would pass into the alimentary tract enabling the peptide to be absorbed across the mucosa, passing into the circulation to powerfully potentiate mealinduced insulin secretion and other extrapancreatic actions through activation of both insulin and GLP-1Rs. Such a mechanism would significantly augment the actions of the creature's own enteroinsular pathway and enhance its ability to store nutrient excess. However, this scenario would require exendin-4 to escape digestion in the gastrointestinal tract, pass into the blood in amounts sufficient to stimulate glucose-dependent insulin release and trigger a powerful anabolic response. Clearly, testing this hypothesis by oral administration of exendin-4 together with a nutrient load to live Gila monsters, followed by blood sampling, would be extremely challenging for us. However, as an alternative and to test bioactivity, we administered exendin-4 orally, together with glucose, to fasted non-diabetic mice and compared the insulin-releasing and glycaemic responses to those induced by glucose given alone or with GLP-1 (Figure 1). We also compared the results with the same dose of exendin-4 given by intraperitoneal injection. As expected, systemic exendin-4 stimulated insulin release (Figure 1B) and significantly moderated the glycaemic excursion (Figure 1A,C). Much to our amazement, similar results were observed with oral exendin-4, although requiring >10-fold higher doses for the same effectiveness. In contrast, oral GLP-1 was completely inactive. The effect persisted whether glucose was given orally or by intraperitoneal injection. We did not have the opportunity to monitor circulating exendin-4 due to limited amount of blood, but one remarkable study has demonstrated that circulating exendin-4 levels, measured by immunoenzymetric assay, increased rapidly by more than 100-fold when the Gila monster devoured small rats. Thus exendin-4 delivered orally, via the saliva in this instance, is absorbed Received: 23 June 2020 Revised: 10 August 2020 Accepted: 12 August 2020