The glucagon-like peptide 1 receptor (GLP-1R) is expressed in many tissues and has been implicated in diverse physiological functions, such as energy homeostasis and cognition. GLP-1 analogs are approved for treatment of type 2 diabetes and are undergoing clinical trials for other disorders, including neurodegenerative diseases. GLP-1 analog therapies maintain chronically high plasma levels of the analog and can lead to loss of spatiotemporal control of GLP-1R activation. To avoid adverse effects associated with current therapies, we characterized positive modulators of GLP-1R signaling. We screened extracts from edible plants using an intracellular cAMP biosensor and GLP-1R endocytosis assays. Ethanol extracts from fenugreek seeds enhanced GLP-1 signaling. These seeds have previously been found to reduce glucose and glycated hemoglobin levels in humans. An active compound (N55) with a novel N-linoleoyl-2-amino-γ-butyrolactone structure was purified from fenugreek seeds. N55 promoted GLP-1-dependent cAMP production and GLP-1R endocytosis in a dose-dependent and saturable manner. N55 specifically enhanced GLP-1 potency more than 40-fold, but not that of exendin 4, to stimulate cAMP production. In contrast to the current allosteric modulators which bind to GLP-1R, N55 binds to GLP-1 peptide and facilitates trypsin-mediated GLP-1 inactivation. These findings identify a new class of modulators of GLP-1R signaling, and suggest that GLP-1 might be a viable target for drug discovery. Our results also highlight a feasible approach for screening bioactive activity of plant extracts.
N55 specifically enhances GLP-1(7-36) amide stimulated cAMP production in RINm5F cells. Effect of increasing concentration of N55 on cAMP production in response to GLP-1(7-36) amide titration in RINm5F cells expressing RG-cAMP biosensor.
N55 enhances GLP-1(7-36) amide dependent GLP-1R endocytosis. Receptor endocytosis assays were performed in U2OS cells co-expression β-arrestin2-GFP and GLP-1R-V2R in the presence of indicated concentration of GLP-1(7-36) amide and N55
N55 enhances GLP-1-elicited insulin release from RINm5F cells.
Student's t test (&*, p< 0.005; ***, p< 0.001) for insulin release by vehicle versus the presence of indicated compounds.
King K, Lin NP, Cheng YH et al., J Biol Chem. 2015 Sep 2. pii: jbc.M115.672097. [Epub ahead of print]