For another the N terminal sequence of native
For another, the N-terminal sequence of native glucagon is highly conserved, and positions 8, 9, 16 and 18 are noteworthy in maintaining glucagon activity , , . It also achieves a degree of selectivity from its C-terminal residues, and a significant contribution is made by the C-terminal Sulfo-NHS-Biotin . In preliminary study, we have studied that the fatty acid side chain helps the molecules to interact with the albumin so that the half-time of GLP-1 could be prolonged to ∼18 h or even longer . And our group contrived a method to modify the peptide rapidly and quantitatively through cysteine , , , maleimide modified fatty acid chain was attached to the cysteine through the thiol group with high chemoselectivity to prolong the half-lives of peptide analogues. Therefore, in the present study, the middle section of glucagon was modified and substituted by cysteine at positions 19–30 to afford twelve glucagon peptide analogues (Fig. 1). A key question is, how the sensitivity of glucagon single amino site on receptor activation and how to find more balanced GLP1-R/GCGR agonist, and even further provide guidance for further GLP-1R/GCGR agonist design. Consequently, through cysteine scanning, 1–12 was synthesized. The GLP-1R/GCGR mediated activation and biological activity in normal ICR mice were comprehensively performed to trace the better regulators of blood glucose and body weight. GLP-1 exists as 30 amino acids, C-terminally amidated peptide (GLP-1(7–36)-NH2), or as the glycine-extended form, GLP-1(7–37). The former is the predominant form in human plasma being , , . In this study, all of peptides are C-terminally amidated peptides to activate GLP-1R more effectively. To prolong the half-lives of peptide analogues, various fatty acid chains with maleimide were added to optimal cysteine modified glucagon analogues to synthesize long-acting compounds. The GLP-1R/GCGR activation and biological activity were also explored. The most potent conjugates with superior hypoglycemic activity and long term effects were identified. In addition, the novel compounds could be used to control blood glucose and reduced body weight in diet induced obese (DIO) mice. Simultaneously, the beneficial influence to liver morphology and overall energy balance were also observed.
Conclusions In the present study, with the specific aims of erasing the likelihood of hyperglycaemia and to remain the inherent catabolic effects through improving GLP-1R activation and deteriorating GCGR activation, optimizing GCGR/GLP-1R activation to develop long-acting therapeutic agents for diet induced obesity, dyslipidemia and diabetes mellitus. We firstly designed and prepared a series of novel cysteine replaced glucagon analogues. The initial GLP-1R/GCGR activation experiments provide direct evidence of the relevant structure-function relationship of glucagon. Compounds 3, 4, 5, 7 and 12 exhibited increased GLP-1R activation and preserved GCGR activation well relative to glucagon, which shows these changes are beneficial. Two positions essential for GLP-1 recognition were found in this study, compounds 10, 11 almost had little activation at GLP-1R, indicating that these residues are sensitive to GLP-1R. Because Phe28 is critical residue for receptor binding and activation on GLP-1R . And compound 10 not only lost its potency at GLP-1R but GCGR, suggesting that this position is important for maintain GLP-1R and GCGR activation. The peptide in vivo results including OGTT and acute food intake and chronic study correlated well with peptides' EC50 and rate of GLP-1R/GCGR activation. Then, various fatty side chain maleimides were added to optimal 4, 5, 7 to prolong peptides lifetime by increased interaction with serum albumin , . Although the negligible reductions were observed in receptor activation, 4d and 7d still showed well-preserved weight-lowering and lipid-regulation effects. Then one-week therapy (the energy balance physiology measurements) and one-month therapy (chronic treatment) in DIO mice further confirmed that 4d had a significant effect in weight loss and energy expenditure increase. Chronic treatment of 4d also lowered body adiposity and hepatic lipid droplets in DIO mice. As expect, the hyperglycemia-promoting effect of 4d is not observed in either one-week or the chronic study, supporting the hypothesis that the increased GLP-1R activation of these molecules opposes and potentially neutralizes GCGR-mediated diabetogenic effects. These preclinical studies indicate that when full GLP-1R activation is augmented with an appropriate degree of GCGR activation, weight reduction can be substantially enhanced without any overt adverse effects. The weight reduction is directly related to the two receptors activation. But how much relative receptor activity is optimal for clinical benefit remains inaccurate and more study is needed.