N‐terminal alterations turn the gut hormone GLP‐2 into an antagonist with gradual loss of GLP‐2 receptor selectivity towards more GLP‐1 receptor interaction

Abstract

Background and Purpose To fully elucidate the regulatory role of the GLP‐2 system in the gut and the bones, potent and selective GLP‐2 receptor (GLP‐2R) antagonists are needed. Searching for antagonist activity, we performed systematic N‐terminal truncations of human GLP‐2(1‐33). Experimental Approach COS‐7 cells were transfected with the human GLP‐2R and assessed for cAMP accumulation or competition binding using 125I‐GLP‐2(1‐33)[M10Y]. To examine selectivity, COS‐7 cells expressing human GLP‐1 or GIP receptors were assessed for cAMP accumulation. Key Results Affinity of the N‐terminally truncated GLP‐2 peptides for the GLP‐2 receptor decreased with reduced N‐terminal peptide length (Ki 6.5–871 nM), while increasing antagonism appeared with inhibitory potencies (IC50) values from 79 to 204 nM for truncation up to GLP‐2(4‐33) and then declined. In contrast, truncation‐dependent increases in intrinsic activity were observed from an Emax of only 20% for GLP‐(2‐33) up to 46% for GLP‐2(6‐33) at 1 μM, followed by a decline. GLP‐2(9‐33) had the highest intrinsic efficacy (Emax 65%) and no antagonistic properties. Moreover, with truncations up to GLP‐2(8‐33), a gradual loss in selectivity for the GLP‐2 receptor appeared with increasing GLP‐1 receptor (GLP‐1R) inhibition (up to 73% at 1 μM). Lipidation of the peptides improved antagonism (IC50 down to 7.9 nM) for both the GLP‐2 and the GLP‐1R. Conclusion and Implications The N‐terminus of GLP‐2 is crucial for GLP‐2R activity and selectivity. Our observations form the basis for the development of tool compounds for further characterization of the GLP‐2 system.

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

“…Because GLP‐2 is a low potency agonist of the GLP‐1R (Gadgaard et al, 2022), and N‐terminal truncations of GLP‐2 result in increased binding to the GLP‐1R (Gabe et al, 2022), we wanted to test how the Ala and Trp substituted GLP‐2 variants affected the selectivity of the peptides and therefore included experiments with the human GLP‐1R. Several variants did not activate the GLP‐1R, but [G4A] , [G4W] , [S5A] , [S5W] , [F6A] , [F6W] and [S7A] did, although with much lower potencies and efficacies than native GLP‐2 (EC 50 of 79 nM and efficacy of 89% relative to GLP‐1 for 1 μM) (Figure 5a‐g).…”

“…A previous Ala scan of GLP‐2[A2G] showed that Ala substitutions in the N‐terminus resulted in less GLP‐2R activation (DaCambra et al, 2000). Furthermore, the truncated metabolite GLP‐2(3‐33) has been shown to be a partial agonist of the GLP‐2R (Gabe et al, 2022; Thulesen et al, 2002). These studies emphasize the importance of the N‐terminus for proper GLP‐2R activation, in line with what has previously been shown for other members of the class B1 family (Gabe et al, 2018; Hansen et al, 2016; Smit et al, 2021; Sparre‐Ulrich et al, 2016; van der Velden et al, 2021).…”

“…These studies emphasize the importance of the N‐terminus for proper GLP‐2R activation, in line with what has previously been shown for other members of the class B1 family (Gabe et al, 2018; Hansen et al, 2016; Smit et al, 2021; Sparre‐Ulrich et al, 2016; van der Velden et al, 2021). In addition, we recently described that removal of the GLP‐2 N‐terminus resulted in lack of GLP‐2R selectivity (Gabe et al, 2022). In contrast, we here find that N‐terminal substitutions of GLP‐2 do not result in impaired GLP‐2R selectivity (Figure 5).…”

Physiological functions of glucose transporter-2: From cell physiology to links with diabetes mellitus

“…The underlying mechanism may be related to GLP-2-dependent activation [ 57 ], where low insulin and high glucagon formed in type 1 diabetes promote GLP-2 secretion in the intestine, increasing GLUT-2 abundance in the BBM. The GLUT-2 transporter performs various other physiological functions, such as stimulating intestinal mucosal growth and resistance to intestinal barrier damage following an inflammatory response [ [58] , [59] , [60] ]. In a type 2 diabetes model, hyperglycemia, hyperinsulinemia, and proglucagon confer an opposite effect due to decreased GLP-2 expression.…”