Fenugreek Compound (N55) Lowers Plasma Glucose through the Enhancement of Response of Physiological Glucagon-like peptide-1.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) analogues are approved for treating type 2 diabetes, but are known to activate GLP-1R signaling globally and constitutively. Active compound N55, previously isolated from fenugreek, enhances the potency of GLP-1 without activating GLP-1R. Here we investigated if N55 lowers plasma glucose base on physiological levels of GLP-1. N55 was found to dose-dependently lower plasma glucose in non-fasted mice but not in the fasted mice, with the effect attenuated by GLP-1R antagonist exendin-(9-39) (Ex-9). On the other hand, when co-administered with dipeptidyl peptidase-IV (DPP4) -resistant [Aib8]-GLP-1(7-36) amide (GLP-1'), hypoglycemic response to N55 was observed in the fasted mice. This enhancement was also found to display dose dependency. N55 enhancement of the hypoglycemic and insulinotropic action of GLP-1' was eliminated upon Ex-9 treatment. Both exendin-4 (Ex-4) and DPP4-resistant GLP-1 mutant peptide ([Aib8, E22, E30]-GLP-1(7-36) amide) activated GLP-1R and improved glucose tolerance but the enhancement effect of N55 was not observed in vivo or in vitro. In conclusions, N55 lowers plasma glucose according to prandial status by enhancing the response of physiological levels of GLP-1 and is much less likely to disrupt tight regulation of GLP-1R signaling as compare to GLP-1 analogues.

Isolation of Positive Modulator of Glucagon-like Peptide-1 Signaling from Trigonella foenum-graecum (Fenugreek) Seed.

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 new 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 that 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.

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

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.

Structural basis for calcium and phosphatidylserine regulation of phospholipase C δ1.

Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.