Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling

Abstract

Glucagon-like peptide-1 (GLP-1) analogs are approved for treatment of type 2 diabetes and are in clinical trials for disorders including neurodegenerative diseases. GLP-1 receptor (GLP-1R) is expressed in many peripheral and neuronal tissues and is activated by circulating GLP-1. Other than food intake, little is known about factors regulating GLP-1 secretion. Given a normally basal circulating level of GLP-1, knowledge of mechanisms regulating GLP-1R signaling, which has diverse functions in extrapancreatic tissues, remains elusive. In this study, we found that the potency of GLP-1, not exendin 4, is specifically enhanced by the endocannabinoid-like lipids oleoylethanolamide (OEA) and 2-oleoylglycerol but not by stearoylethanolamide (SEA) or palmitoylethanolamide. 9.2 μM OEA enhances the potency of GLP-1 in stimulating cAMP production by 10-fold but does not affect its receptor binding affinity. OEA and 2-oleoylglycerol, but not SEA, bind to GLP-1 in a dose-dependent and saturable manner. OEA but not SEA promoted GLP-1(7-36) amide to trypsin inactivation in a dose-dependent and saturable manner. Susceptibility of GLP-1(7-36) amide to trypsin inactivation is increased 40-fold upon binding to OEA but not to SEA. Our findings indicate that OEA binds to GLP-1(7-36) amide and enhances the potency that may result from a conformational change of the peptide. In conclusion, modulating potency of GLP-1 by physiologically regulated endocannabinoid-like lipids allows GLP-1R signaling to be regulated spatiotemporally at a constant basal GLP-1 level.

Keywords: G protein-coupled receptor (GPCR); bioluminescence resonance energy transfer (BRET); endocannabinoid; lipid; signaling.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

Generation of construct to express fusion protein RG-cAMP sensor.A, overlap extension PCR to generate Rluc8-Epac1148–430pcDNA* encoding Epac1 amino acids 148–430. B, generation of Rluc8-Epac1148–881pcDNA* encoding Epac1 amino acids 148–881. C, generation of construct encoding the three-protein fusion of Rluc8-Epac1148–881(T781A,F782A)-GFP2 (RG-cAMP sensor) containing Epac1 sequence encoding amino acids 148–881, where Thr781 and Phe782 in the Epac1 have been changed to alanine.

FIGURE 2.

Detection of intracellular cAMP by RG-cAMP sensor in RINm5F cells.A, schematic diagram of RG-cAMP sensor stably expressed in RINm5F cells and comprising Epac1 (amino acids 148–881 with point mutations T781A and F782A) fused between the Rluc8(1–311) and GFP2(1–239) proteins with Leu-Gly-Leu and Ala-Thr as linkers between Rluc8 and Epac1148–881(T781A,F782A) and GFP2, respectively. B, reduction in BRET ratio in response to titration of the membrane-permeable cAMP analog 8-Br-2′-O-Me-cAMP-AM in RINm5F cells stably expressing the RG-cAMP sensor. C, BRET responses to increasing concentration of adenylyl cyclase activator forskolin in the presence (red squares) or absence (black circles) of 250 μm adenylyl cyclase inhibitor MDL-12330A. Data are means ± S.E. (error bars) of triplicate assays of three independent experiments.

FIGURE 3.

cAMP responses to titration of GLP-1(7–36) amide, GIP, and glucagon in RINm5F cells stably expressing RG-cAMP sensor.A, dose response of cAMP production to titration of GLP-1(7–36) amide in the absence (black circles) or presence (red squares) of 500 nm Ex-9. B, titration of GIP in the absence (black circles) or presence (red squares) of 5 μm GIP(8–42). C, titration of glucagon in the absence (black circles) or presence (red squares) of 100 μm [des-His1,Glu9]glucagon amide. Data are means ± S.E. (error bars) of triplicate assays from three independent experiments.

FIGURE 4.

Effect of OA, linoleic acid (LA), stearic acid (SA), α-linolenic acid (ALA), γ-linolenic acid (γ-LA), SEA, palmitoyl ethanolamide (PEA), and n-oleoyl dopamine (ODA) on cAMP response to the titration of GLP-1(7–36) amide in RINm5F cells. cAMP production in response to the titration of GLP-1(7–36) amide. A, with 106.2 (red squares) and 10.6 μm OA (blue triangles) and vehicle (black circles). B, with 107 (red squares) and 10.7 μm linoleic acid (blue triangles) and vehicle (black circles). C, with the indicated concentration of stearic acid (red squares), α-linolenic acid (blue triangles), γ-linolenic acid (red inverted triangles), and vehicle (black circles). D, with the indicated concentration of SEA (red squares), palmitoylethanolamide (blue triangles), and vehicle only (red diamonds); E, with 72 μmN-oleoyldopamine (red squares) and vehicle only (black circles). Data are means ± S.E. (error bars) of triplicate assays from at least two independent experiments.

FIGURE 5.

OEA and 2-OG enhance GLP-1(7–36) amide-stimulated cAMP productions in RINm5F cells.A and B, cAMP production in response to dose titration of GLP-1(7–36) amide (black circles) alone or in the presence of the indicated concentration of OEA (A) and 2-OG (B). C and D, titration of exendin 4 for cAMP production in the presence of the indicated concentration of OEA (C) and 2-OG (D). E, cAMP production in response to 200 pm GLP-1(7–36) amide alone or in the presence of 500 nm Ex-9 and the indicated concentration of OEA and 2-OG. Data are means ± S.E. (error bars) of triplicate assays from three independent experiments; Student's t test (p < 0.001) for cAMP response elicited by 200 pm GLP-1(7–36) in the absence versus the presence of 2-OG or OEA.

FIGURE 6.

Binding of GLP-1 to OEA, 2-OG, and SEA.A, sequence of His-tagged GLP-1(7–36). Six consecutive histidine residues are tagged to the C terminus of the GLP-1(7–36) peptide. B, binding of 0.2 μm [3H]OEA to increasing concentrations of His-tagged GLP-1(7–36). C, binding of 0.1 μm His-tagged GLP-1(7–36) to increasing concentration of [3H]OEA. D, competition of 2-OG and SEA for the binding of 0.2 μm OEA to 0.2 μm His-tagged GLP-1(7–36). Binding reactions, separation of His-tagged GLP-1(7–36)-bound [3H]OEA and free [3H]OEA, and quantitation of specific bound [3H]OEA are described under “Experimental Procedures.” All data are means ± S.E. (error bars) of triplicate determinations from three independent experiments.

FIGURE 7.

Effect of OEA on saturation binding for GLP-1R. Shown are total (black circles) and nonspecific binding (blue squares) of [125I]GLP-1(7–36) to GLP-1R-V2R membrane in the absence (A) or presence (B) of 9.2 μm OEA. C, specific binding of [125I]GLP-1(7–36) to GLP-1R-V2R membrane in the absence (black circles) or presence (red squares) of 9.2 μm OEA. Binding reactions (220 μl) were carried out in the absence (Total Binding) or presence (Non-specific Binding) of a 500-fold excess of unlabeled exendin 4. Separation of bound and free [125I]GLP-1(7–36) and calculation of specific binding are described under “Experimental Procedures.” Specific binding was determined by subtracting nonspecific binding from total binding. All data are means ± S.E. (error bars) of three independent experiments performed in duplicate. Data were fitted globally to a one-site saturation isotherm.

FIGURE 8.

Effect of OEA on trypsin inactivation of GLP-1(7–36) amide.A, GLP-1(7–36) amide and potential trypsin cleavage fragments, GLP-1(7–26) and GLP-1(7–34). B, cleavage of substrate by 0.000125% trypsin was assayed in the presence (blue squares) and absence (open circles) of 92 μm OEA. The extent of cleavage was monitored by real-time reading of optical density (O.D.) at 405 nm (see “Experimental Procedures”). C and D, cAMP production in response to residual GLP-1(7–36) amide after inactivation by the indicated concentration of trypsin in the absence (C) or presence (D) of 92 μm OEA. E and F, cAMP production in response to residual GLP-1(7–36) amide after inactivation by 0.00067% trypsin in the presence of the indicated concentration of OEA (E) or SEA (F). All of the activity assays were carried out in the presence of 9.2 μm OEA, except in F, where cAMP assays were carried out in the presence of 9.2 μm SEA. All data are means ± S.E. (error bars) of duplicate assays from three independent experiments.