Signalling by the glucagon-like peptide-1 (GLP-1) receptor: interactions between the ago-allosteric agent ‘ compound 2 ’ and the major GLP-1 metabolite, GLP-1 9-36 amide.

J Lu1, NC Li1,2, GB Willars1. 1University of Leicester, Leicester, UK, 2Tianjin Medical University, Tianjin, China

Nutrient ingestion results in release of glucagon-like peptide-1 (GLP-1) from intestinal L-cells. GLP-1 mediates a variety of effects through a Family B GPCR, the GLP-1 receptor (GLP-1R) including the stimulation of glucose-dependent insulin release, making the receptor a target for treatment of type 2 diabetes. GLP-1 7-36 amide (the predominant circulating active GLP-1) is rapidly truncated by dipeptidyl peptidase-4 to GLP-1 9-36 amide, which is generally considered inactive or a low affinity GLP-1R antagonist. This metabolic instability has driven the search for modified peptides or small molecule agonists with therapeutic potential. Amongst the latter, ‘compound 2’ has been described as an agonist and positive allosteric modulator of GLP-1 affinity (Knudsen et al., 2007). Recently we demonstrated that compound 2-mediated cAMP generation is enhanced by exendin 9-36; a GLP-1R peptide antagonist (Coopman et al., 2010). Furthermore, compound 2 and GLP-1 9-36 amide (the major post-prandial circulating GLP-1) synergistically increase cAMP generation by recombinant GLP-1Rs (Li et al., 2012), highlighting that allosteric regulation of metabolites of GPCR ligands may have therapeutic potential. Here we extended these studies to other signalling events and also to signalling by natively expressed GLP-1Rs.

In HEK293Flp-In cells with stable expression of human GLP-1Rs (Coopman et al., 2010), GLP-1 9-36 amide (15min with 500μM isobutylmethylxanthine) increased cAMP (measured by radioreceptor assay) with low potency and low efficacy (pEC50 6.26±0.08, Emax 340±16 with basal 5±2 pmol/mg protein; data are mean±sem, n>3) compared to GLP-1 7-36 amide (10.18±0.11 and 1680±70 respectively). Compound 2-mediated cAMP responses in these cells are bell-shaped (Emax at 3μM ˜70% of GLP-1 7-36 amide; Coopman et al., 2010). Here, co-addition with compound 2 (0.1μM), which alone evoked little or no response, significantly (P<0.001, t test) increased the potency (pEC50 6.49±0.04 vs. 7.49±0.16) and efficacy (Emax at 10µM increased by ˜60%) of the GLP-1 9-36 amide response. Further, GLP-1 9-36 (10μM) or compound 2 (100μM) evoked thapsigargin-sensitive elevations of intracellular [Ca2+] ([Ca2+]i) (measured in fluo-4-loaded cells), which were ˜40% and 50% respectively of those evoked by GLP-1 7-36 amide. Co-addition of concentrations of compound 2 (10µM) and GLP-1 9-36 amide (1µM) that alone evoked little or no Ca2+ signalling evoked robust, thapsigargin-sensitive, elevations of [Ca2+]i. GLP 9-36 amide and compound 2 also activated extracellular signal-regulated kinase (determined by immunoblot of phospho-ERK1/2). Concentrations of GLP 9-36 amide (1µM) and compound 2 (0.1µM) that individually evoked little or no activation of ERK respectively showed responses ˜50% greater than the numerical sum of the individual responses. We were unable to detect GLP-1R-mediated Ca2+ signalling in the pancreatic β-cell line, INS-1E. However, interactions between compound 2 and GLP-1 9-36 amide on cAMP generation and ERK activation seen at the recombinant receptor were present in these cells. Thus, metabolites of peptide hormones, including GLP-1, which may be considered inactive could allow manipulation of receptor function and a novel therapeutic strategy.

Knudsen et al., (2007) Proc Natl Acad Sci USA, 104: 937-942.

Coopman et al., (2010) J Pharmacol Exp Ther, 334: 795-808.

Li et al., (2012) pA2 online 10 (1) 053P