Biased GLP‐2 agonist with strong G protein‐coupling but impaired arrestin recruitment and receptor desensitization enhances intestinal growth in mice

Abstract

Background and Purpose Glucagon‐like peptide‐2 (GLP‐2) is secreted postprandially by enteroendocrine L‐cells and stimulates growth of the gut and bone. One GLP‐2 analogue is approved for short bowel syndrome (SBS). To improve therapeutic efficacy, we developed biased GLP‐2 receptor (GLP‐2R) agonists through N‐terminal modifications. Experimental Approach Variants with Ala and Trp substitutions of the first seven positions of GLP‐2(1‐33) were studied in vitro for affinity, G protein activation (cAMP accumulation), recruitment of β‐arrestin 1 and 2, and internalization of the human and mouse GLP‐2R. The intestinotrophic actions of the most efficacious (cAMP) biased variant were examined in mice. Key Results Ala substitutions had more profound effects than Trp substitutions. For both, alterations at positions 1, 3 and 6 most severely impaired activity. β‐arrestin recruitment was more affected than cAMP accumulation. Among Ala substitutions, [H1A], [D3A] and [F6A] impaired potency (EC50) for cAMP‐accumulation >20‐fold and efficacy (Emax) to 48%–87%, and were unable to recruit arrestins. The Trp substitutions, [A2W], [D3W] and [G4W] were partial agonists (Emax of 46%–59%) with 1.7–12‐fold decreased potencies in cAMP and diminished β‐arrestin recruitment. The biased variants, [F6A], [F6W] and [S7W] induced less GLP‐2R internalization compared with GLP‐2, which induced internalization in a partly arrestin‐independent manner. In mice, [S7W] enhanced gut trophic actions with increased weight of the small intestine, increased villus height and crypt depth compared with GLP‐2. Conclusion and Implications G protein‐biased GLP‐2R agonists with diminished receptor desensitization have superior intestinotrophic effects and may represent improved treatment of intestinal insufficiency including SBS.

Differential Responses of the GLP-1 and GLP-2 Receptors to N-Terminal Modification of a Dual Agonist

“…The affinity trend differs from that previously observed for GLP-1­(7-36) variants with the nLuc-GLP-1R . In the GLP-2-NH 2 series, Gly4 → l -Ala had no effect on receptor affinity (consistent with findings of Gabe et al), and Gly4 → d -Ala caused only a small decline in affinity. In contrast, substantial GLP-1R affinity declines were previously observed for the Gly10 → l -Ala and Gly10 → d -Ala replacements in GLP-1­(7-36) .…”

Computational Design of Potent and Selective d-Peptide Agonists of the Glucagon-like Peptide-2 Receptor

“…The enhancement in the p-AKT levels induced for the d -GLP-2 analogues could be explained by the GLP-2R’s more prolonged activation, given that the d -GLP-2 analogs induce a lower β-arrestin recruitment (Figure D). A recent study showed that biased GLP-2 agonists, with strong G protein-coupling but impaired β-arrestin recruitment and receptor desensitization, enhance intestinal growth in mice . These authors also found that the GLP-2R could be internalized without β-arrestins but with lower efficiency and speed than with the presence of β-arrestins .…”

“…32 These authors also found that the GLP-2R could be internalized without β-arrestins but with lower efficiency and speed than with the presence of β-arrestins. 32 Arrestins are considered to be the central regulators of GPCR endocytosis because they bind to both phosphorylated receptors and adaptor protein 2 (AP-2) or clathrin, attracting receptors to clathrin-coated pits to promote internalization. 33 activation of the GLP-2R compensates for the lower levels of cAMP induced by the D-GLP-2 analogues, making the D-GLP-2 analogues induce similar GFP expression levels as L-GLP-2.…”

“…A recent study showed that biased GLP-2 agonists, with strong G protein-coupling but impaired β-arrestin recruitment and receptor desensitization, enhance intestinal growth in mice . These authors also found that the GLP-2R could be internalized without β-arrestins but with lower efficiency and speed than with the presence of β-arrestins . Arrestins are considered to be the central regulators of GPCR endocytosis because they bind to both phosphorylated receptors and adaptor protein 2 (AP-2) or clathrin, attracting receptors to clathrin-coated pits to promote internalization .…”

Incretins Beyond the Pancreas: Exploring the Impact of GIP and GLP-1/2 on Bone Remodeling

“…These investigations did not reveal any significant correlation between the use of GLP-1R agonists and changes in areal bone mineral density (aBMD), a measure of bone density in specific body areas [30]. Moreover, the administration of GLP-1R agonists did not appear to affect the risk of bone fractures [31,32].…”