Isolation of positive modulator of glucagon-like peptide-1 Signaling from trigonella foenum-graecum (Fenugreek) seed

Article abstract of DOI:10.1074/jbc.M115.672097

Background: Fenugreek is an edible plant used to treat diabetes and other disorders. Results: We isolated a new structure in fenugreek that enhances glucagon-like peptide-1 (GLP-1) potency. Conclusion: The antidiabetic plant fenugreek is a rich source of GLP-1 activity-enhancing compounds. Significance: GLP-1 peptide is a target for new drug screening and drug safety studies.

Full text of DOI:10.1074/jbc.M115.672097

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 43, pp. 26235–26248, October 23, 2015
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Isolation of Positive Modulator of Glucagon-like Peptide-1
Signaling from Trigonella foenum-graecum (Fenugreek)
□
S
Seed*
Received for publication, June 12, 2015, and in revised form, September 1, 2015 Published, JBC Papers in Press, September 2, 2015, DOI 10.1074/jbc.M115.672097
Klim King‡1, Nai-Pin Lin§, Yu-Hong Cheng‡, Gao-Hui Chen¶, and Rong-Jie Chein§2
From the ‡Genomics Research Center, Institutes of §Chemistry and ¶Molecular Biology, Academia Sinica, Taipei 11529, Taiwan
Background: Fenugreek is an edible plant used to treat diabetes and other disorders.
Results: We isolated a new structure in fenugreek that enhances glucagon-like peptide-1 (GLP-1) potency.
Conclusion: The antidiabetic plant fenugreek is a rich source of GLP-1 activity-enhancing compounds.
Significance: GLP-1 peptide is a target for new drug screening and drug safety studies.
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 neuro-
degenerative diseases. GLP-1 analog therapies maintain chron-
ically 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 pos-
itive 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-␥-butyrolactone structure was purified from fenugreek
seeds. N55 promoted GLP-1-dependent cAMP production and
GLP-1R endocytosis in a dose-dependent and saturable man-
ner. N55 specifically enhanced GLP-1 potency more than
40-fold, but not that of exendin 4, to stimulate cAMP produc-
tion. 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 high-
light a feasible approach for screening bioactive activity of plant
extracts.
lungs, heart, kidneys, blood vessels, neurons, and lymphocytes
(1). Mice with GLP-1R gene knock-out or with functionally
deficient GLP-1R signaling exhibit impaired glucose homeosta-
sis, learning, and memory (1). Clinical trials have also targeted
GLP-1 signaling in other conditions, including psoriasis, heart
disease, and neurodegenerative disease (2–5). As such, GLP-1R
signaling might be an ideal research target for the discovery and
development of drugs for the treatment of various conditions
from metabolic disorders to neurodegeneration. The GLP-1R
is a current therapeutic target for treatment of type 2 diabetes
(6, 7).
After food intake, plasma levels of GLP-1 rapidly rise 3–4-
fold, enhancing glucose-dependent insulin secretion, to main-
tain normoglycemia (8, 9). Bioactive forms of GLP-1 contain an
alanine at position 2 and are rapidly degraded by dipeptidyl
peptidase 4 to its inactive forms, GLP-1(9–36)-amide or GLP-
1(9–37), following release from gut L cells. In general, GLP-1 is
quickly secreted after food intake and rapidly cleared by dipep-
tidyl peptidase 4 and the kidneys (10). GLP-1 has a very short
half-life and is sensed by its receptor in a transient and tightly
regulated manner in healthy subjects.
Current therapeutic strategies aim to enhance activation of
GLP-1R in target tissues by maintaining chronically high
plasma levels of GLP-1 analogs (6, 7). This approach is in con-
trast to the natural rapid secretion and clearance of GLP-1, and
it can lead to loss of temporal regulation of GLP-1R signaling.
GLP-1R is present in many different tissues and is involved in
diverse physiological functions (1). It is often the case that local
concentrations of GLP-1, rather than systemic levels, regulate
tissue-specific GLP-1R activation (11). Therefore, constitutive
activation of GLP-1R could result in various off-target effects;
chronically high plasma GLP-1 levels can also lead to loss of
spatial or tissue-specific control of GLP-1R activation. In some
Glucagon-like peptide-1 receptors (GLP-1R)3 are expressed
in a wide variety of tissues, including pancreatic islet ␤-cells,
* This work was supported by Academia Sinica, Taiwan. R.-J. C. and K. K. have conditions, tissue-specific impairment of GLP-1R signaling,
filed a patent application covering chemical compositions and applica-
rather than a global decrease in plasma GLP-1, leads to disease
tions for developing N55 analogs.
pathogenesis, as is the case for type 2 diabetes (12–14). These
□S
This article contains supplemental Figs. S1–S3 and Table S1.
1 To whom correspondence may be addressed. Tel.: 886-921-819-527; E-mail:
bmkxk@ibms.sinica.edu.tw or bmkxk@yahoo.com.
findings indicate that constitutive activation of GLP-1R by
chronically high plasma levels of GLP-1 analogs are likely not
2 To whom correspondence may be addressed. Tel.: 886-2-2789-8526; E-mail:
rjchein@chem.sinica.edu.tw.
3 The abbreviations used are: GLP-1R, glucagon-like peptide-1 receptor;
GPCR, G protein-coupled receptor; BRET, bioluminescence resonance
energy transfer; GIP, gastric inhibitory polypeptide; OEA, oleoyl ethanol-
amide; SEA, stearoyl ethanolamide; Ex-9, exendin 9; Ex-4, exendin 4; EA,
ethyl acetate; Hx, hexane; FE, fenugreek.
OCTOBER 23, 2015•VOLUME 290•NUMBER 43
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
the best therapeutic strategy. Clinical observations also suggest (Ex-9) were synthesized by Genomics BioSci & Technology
that side effects, such as nausea, vomiting, and gastrointestinal (Taipei, Taiwan). Forskolin and G418 were from Sigma. RPMI
distress, may be related to chronically high plasma GLP-1 (15). 1640 tissue culture medium, MEM, fetal bovine serum (FBS),
Concerns about risks for developing pancreatitis and pancre- sodium pyruvate, L-glutamine, HEPES, penicillin/streptomy-
atic malignancies with therapies targeting GLP-1R signaling cin, amphotericin B, gentamicin, phenol red-free MEM, and
have also been raised (16). These findings emphasize that other 0.05% trypsin EDTA were from Gibco. Coelentarazine 400a
strategies should be considered when treating GLP-1R-related was from Gold Biotechnology (St. Louis, MO). The 96-well
disorders.
plates for scanning bioluminescent signals were from Perkin-
To avoid these potentially adverse effects of treatment with Elmer Life Sciences. The black wall clear-bottom 384 micro-
GLP-1 analogs, we searched for positive modulators of GLP-1R plates for receptor endocytosis assay were from Thermo Fisher
signaling that themselves do not activate GLP-1R. Compounds Scientific (Waltham, MA). DNA-binding dye Hoechst 33342
that positively modulate GLP-1/GLP-1R signaling would con- was from Molecular Probes (Eugene, OR). PMSF was from U. S.
trol the degree of activation according to endogenous levels of Biochemicals (Cleveland OH). Oleoyl[9,10-3H]ethanolamide
GLP-1 and are less likely to lead to chronic activation of GLP- ([3H]OEA) was from American Radiolabeled Chemicals Inc.
1R. Furthermore, such compounds would not affect temporal (St. Louis, MO). 125I-Tyr-GLP-1(7–36) (125I-GLP-1(7–36)),
control of receptor activation.
UniFilter-96, GF/C, and Microscint-20 and -40, and the 96-well
To this end, we utilized two independent assay platforms plates for scanning bioluminescent signals were from Perkin-
designed to detect positive modulators of GLP-1R activity from Elmer Life Sciences. Copper-IDA-agarose was from Jena Bio-
crude plant extracts. One assay relates to GLP-1R internaliza- science (Germany).
tion, a common step for activation of most of the G protein-
Determination of Concentrations of GLP-1(7–36)-Amide,
coupled receptors (GPCRs) (17). Because activation of GLP-1R Exendin 4, and Exendin 9—Molar concentrations were deter-
leads to an increase in intracellular cAMP production (18), the mined using the equation whereby peptide concentration
second assay employs a real time intracellular cAMP biosensor (M) ϭ (A280 ϫ -fold dilution)/(a ϫ 1200 ϩ b ϫ 5560); 1200 and
(RG-cAMP sensor) (19), capable of sensing intracellular cAMP 5560 are the molar extinction coefficients for tyrosine and tryp-
production within minutes. These two assays were used in our tophan, respectively. a and b are the number of tyrosine and
initial screening of GLP-1 modulatory activity and for tracking tryptophan residues in the peptide, respectively.
and quantifying such activity during compound purification. In
cAMP Assay—Real time intracellular cAMP assay was per-
screening extracts from edible plants, we identified fenugreek formed as described previously (19). In brief, RINm5F cells sta-
seed extract that potentiates both GLP-1-dependent GLP-1R bly expressing the RG-cAMP protein were seeded at 3 ϫ 104
endocytosis and cAMP response. Fenugreek seeds have tradi- cells/well in 96-well white plates in 0.15 ml of RPMI 1640
tionally been used in Middle Eastern, Indian, Mediterranean, medium containing 400 ␮g/ml G418. The next day, cells were
and Central Asian cuisine. Fenugreek seeds may also help with washed twice with 0.1 ml of phenol red-free MEM containing 5
reducing blood sugar levels in diabetic patients (20). In addi- mM HEPES and incubated in the same medium for 1 h. The
tion, fenugreek seed also displays neuroprotective effects (21) medium was replaced with 90 ␮l of the same medium contain-
and anti-inflammatory properties in animal models (22, 23), ing 1 mg/ml BSA and 5 ␮M DeepBlueC. The whole plate was
these effects may be related to GLP-1R signaling.
immediately loaded onto a SpectraMax Paradigm Detection
The ability to potentiate GLP-1 potency would be a highly Platform equipped with a Dual-Color luminescence detection
desirable characteristic of small molecules for the purpose of cartridge and SoftMax Pro 6.2.2 (Molecular Devices, Sunny-
targeting GLP-1R signaling pathways. The methods we vale, CA) to obtain the background BRET signal based on the
describe here represent important conceptual and operational sequential integration of the luminescence detected at 370–
approaches in discovering class B1 positive modulators of 450 and 500–530 nm over 60–150 s. Each well was then stim-
GPCRs. We report the isolation of an active compound with a ulated by adding 10 ␮l of 10ϫ solutions of peptide and lipids
new structure (N55) from fenugreek seeds. N55 itself does not and 1 mg/ml BSA in phenol red-free MEM containing 5 mM
trigger GLP-1R signaling but instead binds to GLP-1 and HEPES, and BRET signals were obtained immediately under
enhances its potency in stimulating GLP-1R.
identical settings. The BRET ratio is the ratio of light emitted
between 90 and 300 s at 500–530 nm to that emitted at 370–
450 nm. The cAMP response was expressed as a percentage of
Experimental Procedures
Reagents —U2OS cell line stably expressing a ␤-arrestin2: cAMP production and was calculated as 100 ϫ (BRET ratio
GFP fusion protein was obtained from Norak Biosciences (now from 0.01 nM GLP-1(7–36)-amide Ϫ BRET ratio from indicated
Molecular Devices, a part of MDS, Mississauga, Ontario, Can- concentration of peptide with or without lipids)/(BRET ratio
ada). RINm5F cells stably expressing the RG-cAMP protein from 0.01 nM GLP-1(7–36)-amide Ϫ BRET ratio from 250 nM
were described previously (19). Stearoyl ethanolamide (SEA), GLP-1(7–36)-amide). The dose-response curve, maximal
2-oleoylglycerol, and oleoyl ethanolamide (OEA) were pur- response, and concentration of peptide needed to yield half-
chased from Cayman Chemical Co. (Ann Arbor, MI). Peptides maximal response (EC50) were obtained by nonlinear regres-
of GLP-1(7–36)-amide, GLP-1(7–36) with six consecutive his- sion to fit the data to the agonist versus response equation using
tidines linked to the C terminus of GLP-1(7–36) (His-tagged Prism software 5.0 (GraphPad, San Diego). Unless specified, all
GLP-1), and gastric inhibitory polypeptide (GIP) were from cAMP response data are the means Ϯ S.E. from three indepen-
LifeTein (Hillsborough, NJ). Exendin 4 (Ex-4) and exendin 9 dent experiments with triplicate assays.
26236 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 290•NUMBER 43•OCTOBER 23, 2015

Fenugreek Compound Binds to and Enhances GLP-1 Potency
Receptor Endocytosis Assay—U2OS osteosarcoma cell line tagged GLP-1 were carried out as described previously (19).
stably expressing a ␤-arrestin2:GFP fusion protein was Briefly, increasing concentrations of N55 or SEA were incu-
obtained from Norak Biosciences. The pcDNA3 GLP-1R-V2R bated with 0.2 ␮M [3H]OEA in the presence (total binding) or
chimeric construct contains the first 440 amino acids of the absence (nonspecific binding) of 0.2 ␮M His-tagged GLP-1. The
GLP-1R (Met-1 to Thr-440) fused to the last 29 amino acids of reaction was carried out in 50 ␮l of Dulbecco’s phosphate-buff-
the vasopressin V2 receptor (Ala-343 to Ser-371) (24) and sep- ered saline (DPBS) containing 0.02 mg/ml bovine serum albu-
arated by two alanine residues as linker. GLP-1R-V2R chimeric min (BSA) at room temperature for 90 min. Copper-iminodi-
construct is inserted into the EcoRI site of pcDNA3 acetic acid-agarose (Cu2ϩ-agarose), 3 ␮l in 30 ␮l of DPBS, was
(pcDNA3-GLP-1R-V2R) such that expression of the chimeric added to capture all His-tagged GLP-1 (up to 60 ␮M peptide in
protein is under the control of the CMV promoter. an 80-␮l reaction), and the mixture was further incubated with
pcDNA3-GLP-1R-V2R was used to transfect U2OS osteosar- rotation at room temperature for 30 min. The mixture was cen-
coma cells stably expressing ␤-arrestin2:GFP to obtain a cell trifuged at 20,600 ϫ g for 10 min at 4 °C to precipitate His-
line stably co-expressing GLP-1R-V2R and ␤-arrestin2:GFP. tagged GLP-1 trapped in the resin, and 20 ␮l of supernatant
High content imaging of receptor endocytosis in cells was con- containing the unbound free [3H]OEA was mixed with 120 ␮l of
ducted with 0.03 to 0.001 mg/ml extract to identify potentiating MicroScint 40 (PerkinElmer Life Sciences) for quantification of
activity for GLP-1-dependent GLP-1R endocytosis. Extract tritium using single-photon counting (60 s/well read) on a Top-
were supplied at a concentration of 100 mg/ml in 100% DMSO. Count scintillation counter (PerkinElmer Life Sciences). Spe-
Three replicate 384-well assay microplates were plated with cific binding was calculated, wherein bound [3H]OEA ϭ
U2OS cells stably co-expressing GLP-1R-V2R and ␤-arrestin2: (supernatant [3H]OEA of nonspecific binding reaction) Ϫ
GFP at a density of 3 ϫ 103 cells/well. Aliquots of 2.5 ␮l of 10ϫ (supernatant [3H]OEA in the total binding reaction).
stocks of the indicated concentration of extract in phenol red-
Preparation of GLP-1 Receptor Membrane—U2OS cells sta-
free MEM containing increasing concentrations (0.12–3000 bly expressing GLP-1R-V2R were grown to 90% confluence
nM) of GLP-1(7–36)-amide and 1 mg/ml BSA were transferred (about 107 cell per 15 cm dish). The media were removed and
to each well of the assay plate, which contained 22.5 ␮l of phe- washed twice with 30 ml of PBS, followed by adding 2.2 ml of
nol red-free MEM containing 1 mg/ml BSA. The three assay ice-cold homogenization buffer (20 mM HEPES, 1 mM EDTA,
plates were incubated at room temperature for 60 min before 0.7% protease inhibitor mixture P8340 (Sigma) per dish. The
cell fixation with 2% formaldehyde and labeling of the cell cells were scraped immediately and centrifuged at 3000 ϫ g,
nuclei with 5 ␮g/ml of the DNA-binding dye Hoechst 33342 for 4 °C, for 30 min. The pellet was resuspended with 5 ml of
1 h. Plates were washed twice with PBS and sealed; plates were homogenization buffer and then homogenized at maximal
used immediately.
speed on a Polytron 3000 for 10 s on ice with a 30-s interval rest,
Imaging and Analysis—Images were acquired on an XL three times. The homogenized cells were centrifuged for 10 min
model of the ImageXpressꢀ Micro System (Molecular Devices, at 4 °C and 1000 ϫ g. The supernatants were transferred to a
Sunnyvale, CA) and analyzed with MetaXpress High-Content fresh transparent centrifuge tube and centrifuged for 60 min at
Image Acquisition and Analysis Software (Molecular Devices, 4 °C and 55,000 ϫ g. The pellets were resuspended with resus-
Sunnyvale, CA) using the granularity and variable grain analysis pension buffer (20 mM HEPES, 1 mM MgCl2, 0.7% protease
modules. MetaXpress was used to retrieve images using DAPI inhibitor) by passing through a 22-gauge needle one time, a
(to retrieve the blue fluorescent Hoechst 33342-labeled nuclear 25-gauge needle three times, then a 26-gauge needle one time.
images) and FITC (to retrieve the green fluorescent GFP-␤- The protein concentration of the membrane preparation was
arrestin images) filter sets and a Plan Fluor ELWD objective. A determined according to the Qubit fluorometer instructions
20 ϫ 0.45-numerical aperture microscope objective was used (Life Technologies, Inc.). 4–5 ␮g of this membrane yielded
for the imaging; two fields were imaged per well. The number of greater than 5-fold signal/background with 125I-labeled GLP-1
spots, total area covered by spots, average and integrated fluo- at 5 nM in a 0.22-ml assay volume.
rescence intensity of the spots, and nuclear area and fluores-
Assay for Insulin Release from RINm5F Cells—For measuring
cence intensity were logged into the database. Data from abnor- insulin release, RINm5F cells were seeded at 60,000 cells/well in
mal cells for which the values were above threshold (abnormal: a 96-well plate in 0.15 ml of RPMI 1640 medium. The next day,
average nuclear staining intensity Ͻ500; integrated fluores- cells were washed twice with 0.1 ml of phenol red-free MEM (11
cence intensity of area covered by spots Ͻ150) were ruled out mM glucose) containing 5 mM HEPES, pH 7.0, and 1 mg/ml
using Acuity Xpress, followed by selection of the measurement BSA; cells were incubated at the same medium for 1 h. The
method, spot fluorescence integrated intensity to calculate the medium was replaced with 0.05 ml of the same fresh medium
average number of spots per nucleus. Dose-response curve, containing the indicated compound and were incubated for 30
maximal response, and the concentration needed to yield min at 37 °C. Media were withdrawn, centrifuged, and assayed
half-maximal response were obtained using nonlinear for insulin by rat insulin ELISA (Mercodia AB, Uppsala, Swe-
regression to fit the data to the agonist versus response equation den). Each assay was performed in duplicate, and each was
with Prism software 5.0. The extent of receptor endocytosis repeated three times.
response was expressed as percent of that elicited by 750 nM
GLP-1(7–36)-amide.
Cell Viability Test—Cytotoxicity of H460 cells to various
compounds was assessed using PrestoBlueꢀ reagent, which is
Competition Binding Assay—Competition assays to evaluate modified by the reducing environment of the viable cell and
the binding of signal non-enhancing SEA and N55 to His- turns from blue to red in color. In brief, 3000 H460 cells were
OCTOBER 23, 2015•VOLUME 290•NUMBER 43
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
seeded with 0.1 ml of RPMI 1640 medium in each well of a EA) was further fractionated by normal phase silica gel chro-
96-well plate and incubated at 37 °C for 24 h before compound matography. 25 g of FE-E-EA was absorbed onto 79 g of silica
treatment. After a 72-h compound treatment, PrestoBlueTM gel and then chromatographed on a column (4.5 ϫ 43 cm)
cell viability reagent (Invitrogen) was added to each well packed with 370 g of silica gel by stepwise elution with 2500 ml
according to the manufacturer’s protocol. The plates were fur- of 20:80 ethyl acetate/hexane (20% EA/Hx), 2500 ml of 40:60
ther incubated in the dark at 37 °C in 5% CO2 for 10 min, and ethyl acetate/hexane (40% EA/Hx), 2500 ml of 60:40 ethyl ace-
absorbance of all wells was read at both the 570- and 600-nm tate/hexane (60% EA/Hx), 2500 ml of 80:20 ethyl acetate/
wavelengths using automatic enzyme-linked immunosorbent hexane (80% EA/Hx), and 2500 ml of 100% ethyl acetate (100%
assay plate reader (Molecular Devices, Union City, CA). The EA), followed by 2500 ml of methanol/ethyl acetate (50:50; 50%
raw data were normalized and corrected according to the man- EA/Me). Thirty 500-ml fractions were collected, evaporated,
ufacturer’s protocol. All samples were tested in triplicate, and and subjected to activity tests, and fractions with expected
each test was repeated three times. Cell viability was calculated activities were combined. Final purification of the combined F2
using the following formula: percent of 100 ϫ (average cor- fraction and primary structure elucidation of the active com-
rected values of compound treated cells)/(average corrected pound was contracted to AnalytiCon Discovery GmbH (Pots-
values of untreated cells).
dam, Germany). Briefly, 3 g of the partially purified extract with
Receptor Binding Assay—Assays were conducted in 0.22 ml expected activity from normal phase silica gel chromatography
of 50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, and 1 (F2) was further fractionated by preparative reverse phase-
mg/ml BSA containing 3.9 ␮g of GLP-1 receptor membranes HPLC (RP-HPLC) (LiChrospherꢀ select B column 250 ϫ 25
and varying concentrations of 125I-labeled GLP-1(7–36) radio- mm, 10 ␮m). The column was first eluted using a linear gradi-
active ligand with or without 7.7 ␮M N55 and in the absence ent of 50–95% acetonitrile/methanol in water for 57.7 min at a
(total binding) or presence (nonspecific binding) of 1000-fold flow rate of 80 ml/min, followed by elution with 100% acetoni-
excess unlabeled exendin 4. The reactions were incubated for trile/methanol for 10 min. Based on detected peaks (evapora-
90 min. Prior to filtration, an FC 96-well harvest plate (Milli- tive light scattering detector and UV light), the neighboring
pore, MAFC N0B 10; Billerica, MA) was coated with 0.5% poly- similar fractions per fractionation were partially combined to
ethyleneimine for 30 min, then washed with 50 mM HEPES, pH give 72 fractions. The resulting final fractions were evaporated
7.4. 0.1 ml of binding reaction was transferred to the filter plate and subjected to activity tests. Fractions exhibiting potent
and washed 15 times (0.1 ml per well per wash) with ice-cold 25 activity enhancing GLP-1R signaling through GLP-1 were sub-
mM HEPES, pH 7.4, and 50 mM NaCl. The plate was dried fol- jected to structure elucidation.
lowed by adding 30 ␮l of MicroScint 20 (PerkinElmer Life Sci-
Synthesis and Final Structure Characterization of N55—See
ences) per well and, the activity was determined by using single- supplemental material for synthesis and final structure charac-
photon counting (60 s/well read) on a TopCount scintillation terization of N55.
counter (PerkinElmer Life Sciences). Specific bindings were
Results
obtained by subtracting nonspecific binding from total binding.
Dissociation constant (Kd) for GLP-1(7–36)-amide was
Fenugreek Seed Extracts Potentiate GLP-1 Signaling—Initial
obtained using Prism software 5.0 (Graph Pad, La Jolla, CA). extraction and solvent partition of fenugreek seeds is illustrated
Data shown are the means Ϯ S.E. and were duplicated from in Fig. 1. To investigate GLP-1R signaling, we employed ␤-ar-
three independent experiments.
restin2-GFP biosensing technology, based on the observation
In Vitro Assay of Trypsin Activity in the Presence of N55—To that ␤-arrestin binding of an activated receptor is a convergent
determine whether N55 affects trypsin activity, we used the step in GPCR signaling (17). These processes can be visualized
trypsin activity colorimetric test kit (BioVision, Milpitas, CA). (25), allowing for exclusion of false-positive hits. Because
Briefly, 2 ␮l of substrate were added to a well in a 96-well plate GLP-1R activation also leads to an increase in cAMP produc-
containing 48 ␮l of 0.000125% trypsin in the presence or tion (19), the RG-cAMP sensor (an Epac1 cAMP biosensor)
absence of 77 ␮M N55. Reactions were conducted at room tem- (19) was used to monitor real time cAMP production in rat
perature, and the extent of cleavage was monitored by real time insulinoma RINm5F cells (19). This rapid real time cAMP
reading of absorbance at 405 nm.
response assay significantly reduced detection of nonspecific
Limited Trypsin Digestion of GLP-1(7–36)-Amide—Trypsin effects of the extract mixture and allowed us to detect receptor-
digestion was carried out at 37 °C for 30 min in 0.1 ml of phenol specific responses.
red-free MEM containing 5 mM HEPES, pH 7.0, 2 ␮M GLP-1(7–
Ethanol extracts of seeds of fenugreek (FE-E) enhanced the
36)-amide, 2 ϫ 10Ϫ3 to 2.47 ϫ 10Ϫ5% of trypsin (prepared by response to GLP-1(7–36)-amide stimulation. ␤-Arrestin2-GFP
diluting 0.05% trypsin-EDTA), and the indicated concentra- fusion proteins were originally evenly distributed in the cytosol
tions of N55 or SEA. Reaction was terminated by incubating at of U2OS cells co-expressing ␤-arrestin2-GFP and GLP-1R (Fig.
94 °C for 30 min and then at 4 °C for 10 min, followed by addi- 2A). The formation of pit complex ␤-arrestin2-GFP-GLP-1R
tion of PMSF to 1 mM, and 30 min of incubation at room tem- can be visualized when cells were stimulated with 1 nM GLP-
perature. N55 was adjusted to 7.7 ␮M for all the cAMP 1(7–36)-amide. Pit complexes became more prominent when
responses to titration of the GLP-1 in RINm5F cells.
0.1 mg/ml FE-E was included. FE-E alone did not induce for-
Isolation of N55 from Fenugreek Seeds—Ethanol extraction of mation of pits (Fig. 2A). Fig. 2B illustrates the effect of FE-E on
fenugreek seeds and solvent partition of the extracts are out- receptor endocytosis with titration of GLP-1(7–36)-amide.
lined in Fig. 1. Ethyl acetate fraction of solvent partition (FE-E- EC50 of GLP-1(7–36)-amide required to induce half-maximal
26238 JOURNAL OF BIOLOGICAL CHEMISTRY
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
RINm5F cells expressing the RG-cAMP sensor (19) in a dose-
dependent and saturable manner, with an EC50 of 2.8 nM (Fig.
3). In the presence of 0.1 mg/ml FE-E-EA or FE-E-Hx, EC50 was
reduced to 0.18 or 1.67 nM, respectively. The butanol and the
water fractions did not show any activity. A summary of this
step of fractionation is shown in Table 1. Activity of FE-E was
highly enriched in the ethyl acetate fraction (FE-E-EA), whereas
butanol and water fractions displayed little activity.
Isolation and Structural Elucidation of the Active Com-
pound—Because the FE-E-EA fraction potently enhanced
GLP-1R signaling, this fraction (24 g) was chromatographed on
normal phase silica gel. The column was eluted with mixtures of
proportional hexane, ethyl acetate, and methanol of increasing
polarity. As shown in Fig. 4, the FE-E-EA fraction was separated
into 30 fractions, which can be divided into four major groups
according to their ability to potentiate GLP-1-induced GLP-1R
endocytosis (Fig. 4A). The first group (F1) includes fractions 4
and 5; the second group (F2) includes fractions 7–12; the third
group (F3) includes fractions 18–22; and the fourth group (F4)
includes fractions 24–27. In addition to facilitation of GLP-1R
endocytosis, we also examined the effect of these fractions (10
␮g/ml) on cAMP production induced by 1 nM GLP-1. Only
fractions 8–13 significantly potentiated cAMP response
(Fig. 4B).
Fractions 8–13 were combined (FE-E-EA-F2) and subjected
to further purification by reverse phase chromatography to
yield 72 fractions (Fig. 5). To track activity, each fraction (10
FIGURE 1. Ethanol extraction and solvent partition of 2 kg of fenugreek
␮g/ml) was used tested for its ability to enhance the cAMP
response elicited by 1 nM GLP-1. Fractions 39, 42, 52, 55, 58, 60,
and 62 exhibited potent activity in this respect (Fig. 5). Because
seed. The extraction started with extracting 2 kg of dried seed powder of
fenugreek with 20 liters of 95% ethanol for 20 h. The 519 g of concentrated
and dried ethanol extract (FE-E) were further subjected to solvent partition
with n-hexane, ethyl acetate, n-butanol, and water to give 262 g of n-hexane
fraction (FE-E-Hx), 28.7 g of ethyl acetate fraction (FE-E-EA), 46 g of n-butanol fraction 55 had the highest yield and purity, this fraction (N55)
fraction (FE-E-But), and 174 g of water fraction.
was further analyzed using high resolution MS, MS/MS, IR, and
NMR (1H,13C NMR, HH-COSY, HSQC, and HMBC, supple -
response was reduced from 3.8 to 1.7 nM by 0.1 mg/ml FE-E. mental Figs. S1–S3 and Table S1). The NMR assignment of N55
This effect of FE-E is highly dependent on the concentration of is depicted in Fig. 6, A and B, and the absolute configuration was
GLP-1, as it was diminished when GLP-1 concentration was determined by total synthesis as shown in Fig. 6C through
reduced to 0.01 nM (Fig. 2B). FE-E facilitated receptor endocy- amino ester 4, of which the enantiomer was first reported in
tosis elicited by 1 nM GLP-1(7–36)-amide in a dose-dependent 2002 (26). The synthetic N55 is identical in all respects to the
and saturable manner (Fig. 2C). Receptor endocytosis elicited authentic sample isolated from fenugreek. Accordingly, the
by 1 nM GLP-1(7–36)-amide was no more than 10% of that structure of (Ϫ)-N55 was assigned as (9Z,12Z)-N-((3R,4R,5S)-
elicited by 750 nM GLP-1(7–36)-amide. Addition of 0.0037 4,5-dimethyl-2-oxotetrahydrofuran-3-yl)octadeca-9,12-dienamide.
mg/ml FE-E enhanced receptor endocytosis by up to 20%, and
N55 Enhances GLP-1R Signaling—We analyzed the effect of
the response was saturated at 33%, when the concentration of N55 on GLP-1-induced cAMP production in RINm5F cells sta-
FE-E reached 0.03 mg/ml (Fig. 2C). EC50 of FE-E required to bly expressing RG-cAMP (19). GLP-1 induced cAMP produc-
enhance stimulation by 1 nM GLP-1 was 0.0035 mg/ml. FE-E tion in a dose-dependent and saturable manner (Fig. 7A), with
was further subjected to fractionation by solvent partition to an EC50 of 2.4 nM. N55 enhanced the cAMP response to GLP-1
produce hexane (FE-E-Hx), ethyl acetate (FE-E-EA), and buta- and shifted the dose-response curve to the left Ͼ10-fold. N55
nol fractions (FE-E-Bu) (Fig. 1), and their effects on receptor potently reduced the EC50 of GLP-1 by a factor of 40 as its
endocytosis were examined (Fig. 2, D–F). Signal-enhancing concentration increased from 0.8 to 26 ␮M (Fig. 7A). The aug-
activity was enriched in FE-E-EA. EC50 of extract required to mentation diminished as GLP-1 concentration fell to 1 pM,
enhance receptor endocytosis by 1 nM GLP-1 was reduced from indicating a lack of intrinsic agonistic activity of N55 (Fig. 7A).
0.0035 mg/ml (FE-E) to 0.0009 mg/ml (FE-E-EA) (Table 1). N55 increased the potency of GLP-1 in a dose-dependent and
Because GLP-1R activation leads to intracellular cAMP pro- saturable manner (Fig. 7B), with an EC50 of 3.7 ␮M. The EC50 of
duction in pancreatic ␤-cells, we tested whether these extracts GLP-1 decreased from 2.2 to 1.52, 0.96, 0.17, 0.07, and 0.05 nM
potentiate GLP-1-induced cAMP production in RINm5F cells. in the presence of 0.3, 0.8, 2.6, 7.7, and 26 ␮M N55, respectively
We examined the effect of 0.1 mg/ml FE-E-Hx and FE-E-EA (Table 2). These results correspond to 1.45-, 2.3-, 13-, 31-, and
fractions on the potency of GLP-1 stimulation of cAMP pro- 40-fold increases in potency of GLP-1 as the concentration of
duction (Fig. 3). GLP-1 stimulated cAMP production in N55 increased from 0.3 to 26 ␮M. cAMP response to 150 pM
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
FIGURE 2. Effect of extracts from fenugreek seeds on GLP-1 receptor endocytosis to the titration of GLP-1(7–36)-amide. A, fluorescent images of effect
of vehicle, 0.1 mg/ml ethanol extract of fenugreek seed (FE-E), 1 nM GLP-1(7–36)-amide, and 1 nM of GLP-1(7–36)-amide plus 0.1 mg/ml FE-E on the distribution
of ␤-arrestin2-GFP in U2OS cells co-expressing GLP-1R-V2R. B, dose-response curve of receptor endocytosis in response to increasing concentrations of
GLP-1(7–36)-amide in the presence (black triangles) or absence (black circles) of 0.1 mg/ml FE-E. GLP-1R endocytosis elicited by 1 nM GLP-1(7–36)-amide and in
the presence of increasing concentration of FE-E (C), n-hexane partition fraction of FE-E (FE-E-Hx) (D), ethyl acetate partition fraction of FE-E (FE-E-EA) (E) and
n-butanol partition fraction of FE-E (FE-E-Bu) (F). Data are mean Ϯ S.E. from triplicate of three independent experiments.
TABLE 1
Summary of solvent partition of fenugreek seed
Weight
yield
EC50a (mg/ml)
Specific activityb
Total activity
Activity
yield
؊3
Fraction
Weight
؋ 10
(units/mg) ؋ 104
(units) ؋ 109
g
4000
512
262
28.7
46
%
%
FE seed
100
Ethanol extract (FE-E)
25.6
13.1
1.435
2.3
3.5
1.14
1.6
5.851
4.1
100
n-Hexane fraction (FE-Hx)
Ethyl acetate fraction (FE-EA)
n-Butanol fraction (FE-Bu)
H2O fraction (FE-W)
2.55
0.94
24.4
NDc
70.0
20.03
1.29
4.25
0.164
ND
1.22
0.08
173
8.65
a EC50 is the concentration of extract required to achieve half-maximal receptor endocytosis response to the titration of extracts from 0.03 to 100 ␮g/ml in the presence of 1
nM GLP-1(7–36)-amide in an assay volume of 0.025 ml. 1 unit of activity is arbitrarily defined as the activity to the elicited 50% of response in an assay containing 1 nM
GLP-1(7–35)-amide. Data are mean Ϯ S.E. from triplicate of at least two independent experiments.
b Specific activity (units/mg) is arbitrarily defined as the amount of units per mg of extract fraction and is calculated by (1/0.025) ϫ 1/EC50
.
c ND means not done.
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
GLP-1 was enhanced Ͼ5-fold by 7.7 ␮M N55 (Fig. 7C, Student’s induced cAMP response was solely mediated by GLP-1R in
t test, p Ͻ 0.001), and both cAMP response and enhancement
were eliminated by 2 ␮M of the GLP-1R antagonist Ex-9. This
analysis indicates that the enhancing effect of N55 on GLP-1-
RINm5F cells. To examine the specificity of N55 on GLP-1R
signaling, we further analyzed the effect of N55 on cAMP pro-
duction in RINm5F cells stimulated by the gastric inhibitor
polypeptide (GIP). GIP is another incretin, and its receptor is
also expressed in RINm5F cells (27). Activation of the GIP
receptor leads to production of cAMP in RINm5F cells (19, 27).
The cAMP response to either GIP or exendin 4 (Ex-4, a GLP-1
analog) is not affected by N55 (Fig. 7, E and D, respectively).
This analysis indicates that the enhancing effect of N55 on
cAMP production is specific to the GLP-1(7–36)-amide.
N55 Facilitates GLP-1-induced GLP-1R Endocytosis—The
above analyses suggest that through the GLP-1(7–36)-am-
ide, N55 enhances coupling of GLP-1R to G␣s. We further
characterized the effect of N55 on GLP-1R endocytosis elic-
ited by GLP-1(7–36)-amide. EC50 of GLP-1(7–36)-amide-
induced GLP-1R endocytosis was 3.6 nM (Fig. 8A). N55 at a
FIGURE 3. Extracts from fenugreek seed-enhanced GLP-1(7–36)-amide concentration of 7.7 ␮M significantly shifted the dose-re-
stimulated cAMP production in RINm5F cells. cAMP production response
sponse curve of GLP-1 titration to the left and reduced EC50
to GLP-1(7–36)-amide titration in RINm5F cells expressing RG-cAMP sensor
to 0.27 nM. The effects of titration of N55 on GLP-1R endo-
fusion protein in the presence of 0.1 mg/ml hexane partition fraction (FE-E-
Hx) (green squares) or ethyl acetate fraction (FE-E-EA) (red triangles) or vehicle
(black circles). Activity was expressed as cAMP production % of the maximal
response and was calculated as 100ꢁ(A Ϫ B)/(A Ϫ M), where A ϭ BRET ratio by
10 pM GLP-1(7–36)-amide; B ϭ BRET ratio by indicated concentration of GLP-
1(7–36)-amide in the presence of indicated concentration of extract, and M ϭ
BRET ratio by 0.25 ␮M of GLP-1(7–36)-amide. Data are mean Ϯ S.E. of tripli-
cates of three independent experiments.
cytosis elicited by 1 nM GLP-1 indicate that N55 facilitated
GLP-1(7–36)-amide stimulation of receptor endocytosis in a
dose-dependent and saturable manner (Fig. 8B). EC50 of N55
required to enhance receptor endocytosis by 1 nM GLP-1 was
0.87 ␮M.
FIGURE 4. Normal phase silica gel chromatogram of FE ethyl acetate partition fraction. 24 g of FE-E-EA was subjected to normal phase silica gel chroma-
tography and eluted stepwise with solvent of increasing polarity; 20% ethyl acetate in n-hexane (20% EA/Hx), 40% ethyl acetate in n-hexane (40% EA/Hx), 60%
ethyl acetate in n-hexane (60% EA/Hx), 80% ethyl acetate in n-hexane (80% EA/Hx), 100% ethyl acetate (100% EA), and 50% methanol in ethyl acetate (50%
EA/Me) as described under “Experimental Procedures.” A, activity was assayed for each fraction (0.01 mg/ml each fraction was tested for its ability to potentiate
receptor endocytosis elicited by 1 nM GLP-1) and was expressed as % of the response by 0.75 ␮M of GLP-1(7–36)-amide. B, increased cAMP production in
RINm5F cells stably expresses RG-cAMP sensor. Activity was expressed as increased cAMP production (% of the maximal response) and was calculated as
100ϫ(A Ϫ B)/(C Ϫ M), where A ϭ BRET ratio by 1 nM GLP-1(7–36)-amide; B ϭ BRET ratio by 1 nM GLP-1(7–36)-amide in the presence of 0.01 mg/ml indicated
fraction;CϭBRETratioby10pM GLP-1(7–36)-amide, andMϭBRETratioby0.25␮M GLP-1(7–36)-amide. Blacktrianglesandreddiamondsrepresentactivityand
weight of each fraction, respectively.
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
FIGURE 5. Reverse phase chromatography profile of FE-EA-F2 versus activity to enhance cAMP production by GLP-1 in RINm5F cells stably
expressing RG-cAMP sensor. 3 g of FE-EA-F2 were fractionated by preparative RP-HPLC (LiChrospherꢂ select B column 250 ϫ 25 mm, 10 ␮m) and
resolved into 72 fractions. Each fraction (0.01 mg/ml) was tested for its ability to enhance cAMP production by 1 nM GLP-1(7–36)-amide in RINm5F cells
stably expressing RG-cAMP. Activity was expressed as increased cAMP production (% of the maximal response) and was calculated as 100ϫ(A Ϫ B)/(C Ϫ
M), where A ϭ BRET ratio by 1 nM GLP-1(7–36)-amide; B ϭ BRET ratio by 1 nM GLP-1(7–36)-amide in the presence of 0.01 mg/ml indicated fraction; C ϭ
BRET ratio by 10 pM GLP-1(7–36)-amide; and M ϭ BRET ratio by 0.25 ␮M GLP-1(7–36)-amide. Black diamonds represent activity and red triangles indicate
weight (mg) for each fraction.
N55 Facilitates GLP-1-elicited Insulin Release from RINm5F enhanced binding affinity of GLP-1(7–36)-amide to GLP-1R.
Cells—One major physiological response to the activation of We performed saturation binding assays to examine the effects
the GLP-1 receptor in pancreatic ␤-cell lines is insulin release of N55 on GLP-1R binding affinity. Binding of 125I-labeled
(1). As N55 enhanced the potency of GLP-1(7–36)-amide, we GLP-1(7–36)-amide to GLP-1R is dose-dependent and satura-
further examined whether N55 could enhance GLP-1-elicited ble, the dissociation constant (Kd) and maximal binding (Bmax
)
insulin release from RINm5F cell, a rat insulinoma cell line that being 1.5 Ϯ 0.25 nM and 51 Ϯ 2.9 pmol/mg membrane, respec-
has been show to release insulin in response to GLP-1 stimula- tively (Fig. 11A). N55 at a concentration of 7.7 ␮M enhanced
tion (28). As shown in Fig. 9, 2 nM GLP-1(7–36)-amide consis- cAMP response 30-fold, although this concentration of N55
tently stimulated a low level of insulin release from RINm5F barely affected the saturation binding curve (Fig. 11B). Kd and
cells, and this response is significantly enhanced by 7.7 ␮M N55,
Bmax values in the presence of 7.7 ␮M N55 was 1.4 Ϯ 0.5 nM and
although N55 alone does not trigger insulin release. Intracellu- 51 Ϯ 6.5 pmol/mg of membrane, respectively. Specific binding
lar cAMP level is primarily responsible for enhanced glucose- of 125I-labeled GLP-1(7–36)-amide to GLP-1R in the presence
dependent insulin secretion from pancreatic ␤-cells (29). or absence of 7.7 ␮M N55 is almost superimposed (Fig. 11C).
Increasing intracellular cAMP level by forskolin will lead to This analysis suggests that N55 minimally affected GLP-1’s
insulin release from RINm5F cells (Fig. 9). This is consistent affinity for and capacity to bind GLP-1R. Though enhancing the
with the enhancement effect of N55 on cAMP production potency of GLP-1(7–36)-amide in stimulating the cAMP
results from GLP-1 stimulating RINm5F cells.
response, 7.7 ␮M N55 did not affect the physical affinity
N55 Does Not Affect Cell Viability—N55 alone does not affect between GLP-1 and its cognate receptor.
receptor endocytosis, cAMP production, and insulin secretion.
N55 Binds to GLP-1(7–36)-Amide and Facilitates Trypsin
To examine whether these negative effects could result from Inactivation of GLP-1(7–36)-Amide—N55 selectively en-
the cytotoxicity of N55, we compared the effect of N55 on cell hanced the potency of GLP-1(7–36)-amide, but not that of
viability to those of endocannabinoid-like lipids (2-oleoylglyc- Ex-4, possibly because N55 might interact with the GLP-1(7–
erol and oleoylethanolamide) and cisplatin (an anti-apoptosis 36)-amide peptide. It has been shown that GLP-1 binds to sig-
compound). Within the test concentration from 0.78 to 100 nal-enhancing OEA or 2-oleoylglycerol but not to SEA (19).
␮M, N55, 2-oleoylglycerol, and oleoylethanolamide do not Competition binding experiments were performed to examine
show detectable cytotoxicity; however, cisplatin potently killed the effect of increasing N55 concentrations on the binding of
the cell at a concentration of 50 ␮M and displayed an IC50 of 6.7 [3H]OEA to GLP-1. As shown in Fig. 12A, binding of OEA grad-
␮M (Fig. 10). This analysis revealed that N55 displayed compa- ually decreased as the concentration of N55 increased from
rable effects on cell viability as those of endocannabinoid-like 0.025 to 1.5 ␮M, and the inhibition constant (Ki) was estimated
lipids.
to be 0.03 Ϯ 0.01 ␮M. In contrast, SEA did not affect the binding
Effect of N55 on Saturation Binding of GLP-1 to GLP-1R— of OEA to GLP-1. A potential consequence of the binding of
Because N55 enhanced the potency of GLP-1(7–36)-amide, we N55 to GLP-1 is induction of a conformational change in the
further investigated whether this effect may have been due to peptide. It has previously been demonstrated that there are sig-
26242 JOURNAL OF BIOLOGICAL CHEMISTRY
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
FIGURE 6. Structure elucidation of N55. A, structure of N55. B, NMR assignment of N55. C, total synthesis of N55 for the absolute structure confirmation. PMP,
para-methoxyphenyl; CAN, ceric ammonium nitrate; Bn, benzyl; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; DIPEA,
diisopropylethylamine.
nificant concentration-dependent conformational changes in out in the presence of 77 ␮M N55 (Fig. 12C), the residual GLP-1
GLP-1 within the range of 3–50 ␮M (30). Therefore, we utilized activity was dramatically reduced by a factor of Ͼ300 (Fig. 12C).
susceptibility to trypsin digestion to explore potential confor- We also compared the effect of SEA, which was shown to have
mational changes in GLP-1(7–36)-amide at low micromolar no effect on the potency of GLP-1 in stimulating cAMP produc-
concentrations upon binding N55. The two trypsin cleavage tion. Fig. 12D shows that residual GLP-1 activity was reduced
products of GLP-1(7–36)-amide correspond to an inactive only by a factor of 8 in the presence of 92 ␮M SEA. These results
GLP-1 (7–26) and a partially active GLP-1 (7–34) (31). Residual illustrate that trypsin digestion of GLP-1(7–36)-amide was
activity simulating cAMP production after trypsin treatment facilitated by N55, but N55 did not affect innate enzymatic
was used to determine susceptibility of the peptide to trypsin activity of trypsin (Fig. 13). To determine whether this effect of
digestion. Increasing the concentration of trypsin from 2.47 ϫ N55 is dose-dependent, inactivation of GLP-1(7–36)-amide by
10Ϫ5 to 2 ϫ 10Ϫ3% reduced residual GLP-1 activity by a factor of 6.7 ϫ 10Ϫ4% trypsin was carried out with increasing concentra-
27 (Fig. 12B). When the trypsin cleavage reactions were carried tions of N55 from 0.8 to 77 ␮M. Residual activity of GLP-1(7–
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
FIGURE 7. N55 specifically enhances GLP-1(7–36)-amide-stimulated cAMP production in RINm5F cells. A, effect of increasing concentrations of N55 on
cAMP production in response to GLP-1(7–36)-amide titration in RINm5F cells expressing RG-cAMP biosensor. B, N55 dose-dependently and saturably increases
the potency of GLP-1 to stimulate cAMP production. C, effect of exendin 9 on cAMP production in response to 150 pM GLP-1(7–36)-amide alone or together
with the presence of 7.7 ␮M N55. Effect of N55 on cAMP production responses to the titrations of Ex-4 (D) or GIP (E). Activity was expressed as cAMP production
% of the maximal response and was calculated as 100ϫ(A Ϫ B)/(A Ϫ M), where A ϭ BRET ratio by 10 pM GLP-1(7–36)-amide; B ϭ BRET ratio by indicated
concentration of GLP-1(7–36)-amide in the presence of indicated concentration of N55, and M ϭ BRET ratio by 0.25 ␮M GLP-1(7–36)-amide. Increase in potency
of GLP-1 to stimulate cAMP response is calculated by (EC50 of GLP-1)/(EC50 of GLP-1 in the presence of indicated concentration of N55). Data are mean Ϯ S.E.
of triplicates of three independent experiments. Student’s t test (p Ͻ 0.001) for cAMP response elicited by 150 pM GLP-1(7–36) in the absence versus the
presence of N55.
TABLE 2
Effect of increasing concentrations of N55 on EC50 of GLP-1(7–36)-amide to induce cAMP production
N55 concentration (␮M)
0
26
7.7
2.6
0.77
0.26
EC50 (nM)a
2.20 Ϯ 0.64
0.05 Ϯ 0.01
0.07 Ϯ 0.02
0.17 Ϯ 0.04
0.96 Ϯ 0.16
1.52 Ϯ 0.25
a Data are mean Ϯ S.D. from triplicate of at least three independent experiments.
trypsin inactivation (Fig. 12F), with an EC50 of 2 ␮M. These
findings suggest that a conformational transition in the GLP-
1(7–36)-amide peptide occurred upon binding N55.
Discussion
Fenugreek seeds have been used as a treatment for diabetes
and have been found to lower blood glucose levels in human
trials (20, 32–34). We report the isolation and characterization
of a new structure from fenugreek seeds. N55 binds to GLP-1
and enhances the potency of GLP-1 in stimulating GLP-1R sig-
naling. This finding is consistent with research targeting
FIGURE 8. N55 enhances GLP-1(7–36)-amide- dependent GLP-1R endocy-
tosis. Receptor endocytosis assays were performed in U2OS cells co-expres-
sion ␤-arrestin2-GFP and GLP-1R-V2R in the presence of indicated concentra- GLP-1R signaling in the treatment of diabetes. Using agonist-
tions of GLP-1(7–36)-amide and N55. A, response of GLP-1R endocytosis to
induced receptor endocytosis and real time measurement of
thetitrationofGLP-1intheabsence(blackcircles)orpresence(greentriangles)
intracellular cAMP production, we purified and isolated N55
of 7.7 ␮M N55. B, N55 dose-dependently and saturably enhances GLP-1R
endocytosis elicited by 1 nM GLP-1(7–36)-amide. All data are mean Ϯ S.E. of
triplicates from three independent experiments.
from fenugreek seeds. It is noted that although some fractions
from silica gel chromatography did not potentiate cAMP pro-
duction, they were still able to enhance receptor endocytosis
(Fig. 4, A and B). Because cAMP is essential and sufficient for
pancreatic ␤-cell secretion of insulin (29), we combined frac-
tions with enhancing effects on cAMP production for further
36)-amide decreased by a factor of Ͼ30 (Fig. 6E) as the concen-
tration of N55 increased from 0.8 to 77 ␮M. The effect of N55
became saturated as it reached a concentration of 7.7 ␮M (Fig.
12E). These analyses indicate that N55 dose-dependently and
saturably increased susceptibility of GLP-1(7–36)-amide to purification. As shown in Fig. 5, most of the fractions at this step
26244 JOURNAL OF BIOLOGICAL CHEMISTRY
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
dence favor the interpretation that N55 interacts with GLP-
1(7–36)-amide. First, N55 selectively enhanced the potency of
GLP-1(7–36)-amide but not that of Ex-4 (Fig. 7, A and D).
GLP-1 and exendin 4 share 50% sequence identity but have
distinct secondary structure, order, and stability characteristics
(30, 35). Second, N55, but not SEA (19), binds GLP-1 in a dose-
dependent manner (Fig. 12A). Finally, N55 specifically
enhanced susceptibility of the peptide to cleavage by trypsin
(Fig. 12, C and E), although SEA (19) did not (Fig. 12D). These
are consistent with previous findings that only specific signal-
ing enhancing lipids bind GLP-1, while also enhancing its sus-
ceptibility to trypsin digestion (19). These observations are con-
sistent to the model that GLP-1 can bind to specific lipids to
form a complex with enhanced potency in stimulating GLP-1R
signaling. Covalent linking of a lipid molecule to peptide hor-
mones (lipidation) is commonly used as a chemical tool to opti-
mize the pharmacokinetic properties of peptide drugs (36).
Moreover, lipidation may stabilize a structure of the peptide
hormone, leading to broadening its spectrum of activity (37).
Most of the lipidations of GLP-1 have no discernible effects on
FIGURE 9. N55 enhances GLP-1-elicited insulin release from RINm5F cells.
Effects of various compounds on insulin release from RINm5F cells are shown.
Stimulation of RINm5F cells and assays for insulin release were described
under “Experimental Procedures.” Data are means Ϯ S.E. (error bars) of dupli-
cate assays from three independent experiments. Student’s t test (ꢃ, p Ͻ
0.005; ꢃꢃꢃ, p Ͻ 0.001) for insulin release by vehicle versus the presence of
indicated compounds.
GLP-1R signaling, with a few exceptions, which enhanced the
potency of GLP-1 by 6–7-fold (38). In contrast to covalent link-
ing, our studies show that non-covalent binding of N55 to
GLP-1 also stabilized a peptide structure displaying a 40-fold
enhancement in potency to stimulate cAMP production.
The postulated mechanism by which N55 enhances GLP-1R
signaling is different from that of allosteric modulators isolated
in the past (39–42). All previously isolated GLP-1R modulators
exert their effects by binding to allosteric sites on the receptor
and display ago-allosteric modulation. These allosteric modu-
lators display both allosteric agonism and an ability to alloster-
ically modulate the binding, specificity, and/or function of the
GLP-1 ligand. This effect may be due to the allosteric modula-
tor causing a redistribution of conformationally linked receptor
states, which can include signaling species. In contrast, N55 had
a pharmacological profile distinct from that of GLP-1 modula-
tors identified in the past; N55 does not display agonism or
altered receptor specificity in the absence of GLP-1 ligand.
Fenugreek has been used for the treatment of diabetes in
many parts of the world, particularly in China, Egypt, India, and
Middle Eastern countries (43, 44). In humans, fenugreek seeds
acutely reduce post-prandial glucose and insulin levels (32).
Several longer term clinical trials have also shown that admin-
istration of fenugreek seeds reduces fasting and post-prandial
FIGURE 10. Cytotoxicity test of N55. Dose-dependent N55 (red circles),
oleoylethanolamide (green triangles), 2-oleoylglycerol (blue squares), and cis-
platin (black inverted triangles) on the viability of H460 cells. Cell viability
assays are described under “Experimental Procedures.” Data are means Ϯ S.E.
(error bars) of the triplicate assays from three independent experiments.
of isolation enhanced GLP-1-induced cAMP production, glucose levels and decreases glycated hemoglobin (33). Soluble
although fraction N55 is of high recovery and significantly pure. fiber (45, 46), saponins (47), trigonelline (48), diosgenin (49),
The effect of N55 on GLP-1R signaling is specific to the GLP- and 4-hydroxyisoleucine (50) from fenugreek seeds have been
1(7–36)-amide. It does not enhance signaling by the GLP-1 suggested to mediate its hypoglycemic effect (33). The exact
analog Ex-4 (Fig. 7D) or the other incretin GIP (Fig. 7E). N55 molecular targets and mechanisms remain unknown; however,
does not activate GLP-1R by itself, as its effect is highly depen- N55 from fenugreek may potentially play a role in ameliorating
dent upon the presence of GLP-1(7–36)-amide (Fig. 7A). N55 glucose excursion in diabetes. In addition to hypoglycemic
enhanced the potency of GLP-1 stimulation of the cAMP effect, extract of fenugreek seed also displays neuroprotective
response mediated by GLP-1R signaling, and this augmentation properties (21) and anti-inflammatory effects (22, 23) in disease
is eliminated when GLP-1R signaling in RINm5F cells is antag- animal models. These mechanisms of effects are consistent
onized by Ex-9 (Fig. 7C).
with the current trials of GLP-1 analogs in treatment of psori-
There are two possible mechanisms that may explain the asis (5) and Parkinson disease (2).
effect of N55 on GLP-1R signaling. N55 can either act upon
Current GLP-1 analog therapies maintain constant high lev-
GLP-1R or on GLP-1(7–36)-amide. At least three lines of evi- els of GLP-1 activity, leading to constitutive activation of
OCTOBER 23, 2015•VOLUME 290•NUMBER 43
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Fenugreek Compound Binds to and Enhances GLP-1 Potency
FIGURE 11. N55 does not affect the binding of GLP-1(7–36)-amide to GLP-1R. Total (black circles) and nonspecific (black squares) binding of 125I-labeled
GLP-1(7–36) to GLP-1R-V2R membrane in the absence (A) or presence (B) of 7.7 ␮M N55., specific binding of 125I-labeled GLP-1(7–36) to GLP-1R-V2R membrane
in the absence (black circles) or presence (black squares) of 7.7 ␮M N55. Specific bindings are obtained by subtracting nonspecific bindings from total bindings.
All data are mean Ϯ S.E. of duplicates from three independent experiments.
FIGURE 13. Trypsin cleavage activity is not affected by N55. To examine
whetherthepresenceofN55affectstrypsincleavageactivity, weusedtrypsin
activity colorimetric test (BioVision). Briefly, trypsin activity was assayed with
and without 77 ␮M N55; 2 ␮l of trypsin substrate was added to a well in a
96-well plate containing 48 ␮l of 0.000125% trypsin in the presence (blue
squares) or absence (open circles) of 77 ␮M of N55. Reactions were conducted
at room temperature, and the extent of cleavage was monitored by real time
reading at A405
.
GLP-1R and loss of spatiotemporal regulation of GLP-1R activ-
ity. Positive modulation of GLP-1R signaling may offer the
opportunity to augment and/or fine-tune endogenous GLP-1
action. Enhancers of GLP-1 potency modulate GLP-1R signal-
ing according to endogenous levels of GLP-1. They do not
themselves activate the receptor, but serve only to augment the
potency of circulating GLP-1. This property may be of particu-
lar importance for brain GLP-1R-expressing neurons, where
activation of the receptor is regulated by local GLP-1-secreting
fibers.
Deterioration in GLP-1 signaling has been associated with
type 2 diabetes (13). Such patients have impaired incretin func-
tion, despite having normal or only mildly reduced GLP-1
secretion, as measured by oral glucose or meal tests (13, 14).
Clinically, administration of a GLP-1 analog has been success-
ful in ameliorating the effects of reduced GLP-1 signaling in
type 2 diabetes (6, 7). Although the GLP-1 analog therapies may
lead to loss of spatiotemporal regulation of most of GLP-1Rs,
similar therapeutic benefits can be achieved with modulators of
GLP-1 potency. Furthermore, because of its clean modulation
without intrinsic agonist activity, the potential adverse effects
are minimized. This mode of action permits control over the
intensity of activation based on endogenous levels of GLP-1 and
could potentially restore GLP-1 signaling function in a manner
more consistent with physiological demands. As cAMP is suf-
FIGURE 12. N55 binds and facilitates inactivation of GLP-1(7–36)-amide by
trypsin. A, competition of increasing concentrations of SEA or N55 for the bind-
ing of 0.2 ␮M [3H]OEA to 0.2 ␮M His-tagged GLP-1. Binding reactions, separation
ofHis-taggedGLP-1(7–36)-bound[3H]OEA,andfree[3H]OEAquantitationofspe-
cific bound [3H]OEA were described under “Experimental Procedures.” B–D,
cAMPproductionresponsestotitrationsofresidualactivitiesofGLP-1(7–36)after
treatment with the indicated concentrations of trypsin in the presence of vehicle
(B), 77 ␮M N55 (C), or 92 ␮M SEA (D). E, responses of cAMP production to titrations
ofGLP-1(7–36)-amideaftertreatedwith0.00067%trypsininthepresenceofindi-
cated concentrations of N55. F, N55 dose-dependently and saturably facilitates
inactivationofGLP-1(7–36)-amideby0.00067%trypsin. Decreaseinpotencywas
defined by (EC50 of GLP-1 trypsinized in the presence of the indicated concentra-
tion ofN55)/(EC50 of GLP-1trypsinizedin the absence of N55). Except forD, cAMP
responseswereassayedinthepresenceof9.2␮M SEA;allothercAMPproduction
assays were carried in the presence of 7.7 ␮M N55, and all data are mean ϮS.E. of
triplicates from three independent assays.
26246 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 290•NUMBER 43•OCTOBER 23, 2015

Fenugreek Compound Binds to and Enhances GLP-1 Potency
8. Drucker, D. J. (2006) The biology of incretin hormones. Cell Metab. 3,
153–165
ficient for glucose-dependent insulin release (29), future stud-
ies may investigate whether compounds that enhance GLP-1-
induced cAMP production represent a new class of therapeutic
agents for the treatment of type 2 diabetes or other GLP-1 sig-
naling-related disorders. To this end, examination of in vivo
effects of N55 is necessary. Other studies may also investigate
the viability of nutrient derivatives from edible plants in treat-
ing or preventing type 2 diabetes or other GLP-1 signaling-
related conditions.
9. Meier, J. J. (2012) GLP-1 receptor agonists for individualized treatment of
type 2 diabetes mellitus. Nat. Rev. Endocrinol. 8, 728–742
10. Meier, J. J., Nauck, M. A., Kranz, D., Holst, J. J., Deacon, C. F., Gaeckler, D.,
Schmidt, W. E., and Gallwitz, B. (2004) Secretion, degradation, and elim-
ination of glucagon-like peptide 1 and gastric inhibitory polypeptide in
patients with chronic renal insufficiency and healthy control subjects.
Diabetes 53, 654–662
11. Gu, G., Roland, B., Tomaselli, K., Dolman, C. S., Lowe, C., and Heilig, J. S.
(2013) Glucagon-like peptide-1 in the rat brain: distribution of expression
and functional implication. J. Comp. Neurol. 521, 2235–2261
12. Nauck, M. A., Vardarli, I., Deacon, C. F., Holst, J. J., and Meier, J. J. (2011)
Secretion of glucagon-like peptide-1 (GLP-1) in type 2 diabetes: what is
up, what is down? Diabetologia 54, 10–18
In summary, we identified a new compound from fenugreek
seeds with potential clinical application in the treatment of type
2 diabetes and other GLP-1 signaling-related disorders. N55
binds to GLP-1(7–36)-amide, enhances the potency in stimu-
lating the cAMP pathway. This mechanism is distinct from that
of classical GLP-1R-signaling agents, which directly act on the
target receptor orthosterically or allosterically. N55 thus repre-
sents a new class of compounds that enhance GLP-1R signaling.
Our results also suggest that GLP-1 might be a novel target for
drug discovery in type 2 diabetes and other conditions. The
postulated mechanism of N55 action also points to the possi-
bility of modulating GPCR activity through modification of the
potency of its cognate ligands. The approach described here is
applicable to many other plant compound discovery efforts
related to other GPCRs and their respective ligands.
13. Calanna, S., Christensen, M., Holst, J. J., Laferrère, B., Gluud, L. L., Vilsbøll,
T., and Knop, F. K. (2013) Secretion of glucagon-like peptide-1 in patients
with type 2 diabetes mellitus: systematic review and meta-analyses of clin-
ical studies. Diabetologia 56, 965–972
14. Færch, K., Torekov, S. S., Vistisen, D., Johansen, N. B., Witte, D. R., Jons-
son, A., Pedersen, O., Hansen, T., Lauritzen, T., Sandbæk, A., Holst, J. J.,
and Jørgensen, M. E. (2015) GLP-1 response to oral glucose is reduced in
prediabetes, screen-detected type 2 diabetes, and obesity and influenced
by sex: The ADDITION-PRO Study. Diabetes 64, 2513–2525
15. Lovshin, J. A., and Drucker, D. J. (2009) Incretin-based therapies for type 2
diabetes mellitus. Nat. Rev. Endocrinol. 5, 262–269
16. Butler, P. C., Elashoff, M., Elashoff, R., and Gale, E. A. (2013) A critical
analysis of the clinical use of incretin-based therapies: Are the GLP-1
therapies safe? Diabetes Care 36, 2118–2125
17. Pierce, K. L., and Lefkowitz, R. J. (2001) Classical and new roles of ␤-ar-
restins in the regulation of G-protein-coupled receptors. Nat. Rev. Neuro-
sci. 2, 727–733
Author Contributions—R.-J. C. designed the synthetic plan and syn-
thesized and assigned the structure of N55. K. K. set up the signaling
assay platforms, isolated N55, and designed biological and pharma-
cological characterization of N55. Y.-H. C. performed the cAMP
BRET assays, and ligand binding assays. N.-P. L. implemented syn-
thetic experiments and spectrometry analysis. Receptor endocytosis
assays were done by G.-H. C. Y.-H. C. and K. K. set up the cAMP
assay platform. R.-J. C. and K. K. wrote the paper.
18. Widmann, C., Bürki, E., Dolci, W., and Thorens, B. (1994) Signal trans-
duction by the cloned glucagon-like peptide-1 receptor: comparison with
signaling by the endogenous receptors of beta cell lines. Mol. Pharmacol.
45, 1029–1035
19. Cheng, Y. H., Ho, M. S., Huang, W. T., Chou, Y. T., and King, K. (2015)
Modulation of glucagon-like peptide-1 (GLP-1) potency by endocannabi-
noid-like lipids represents a novel mode of regulating GLP-1 receptor
signaling. J. Biol. Chem. 290, 14302–14313
Acknowledgments—We thank Dr. Mei-Shang Ho, Dr. Lee-Young
Chau, and Dr. Chi-Huy Wong for constructive discussions.
20. Haber, S. L., and Keonavong, J. (2013) Fenugreek use in patients with
diabetes mellitus. Am. J. Health Syst. Pharm. 70, 1196
21. Belaïd-Nouira, Y., Bakhta, H., Samoud, S., Trimech, M., Haouas, Z., and
Ben Cheikh, H. (2013) A novel insight on chronic AlCl3 neurotoxicity
through IL-6 and GFAP expressions: modulating effect of functional food
fenugreek seeds. Nutr. Neurosci. 16, 218–224
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Supplemental Information
Isolation of Glucagon-like Peptide-1 Signaling Positive Modulator from Seed of
Trigonella foenum-graecum (Fenugreek)
Klim King, Nai-Pin Lin, Yu-Hong Cheng, Gao-Huei Chen, and Rong-Jie Chein
General Experimental Methods
All reactions were carried out under an inert nitrogen atmosphere with dry solvents under
anhydrous conditions unless otherwise stated, and standard syringe–septa techniques were
followed. Solvents were dried by conventional methods prior to use. Reagents were
purchased and used without further purification. Reactions were monitored by thin-layer
chromatography (TLC) using glass-baked plates pre-coated with silica gel 60 (Merck, silica
gel 60 F254). Ultraviolet (UV) light was used as the visualizing agent. Ceric ammonium
molybdate and heat, ninhydrin and heat or iodine were used as developing agents. Flash silica
gel chromatography was performed using silica gel 60 (Merck, F254 230–400 mesh). Nuclear
magnetic resonance (NMR) spectra were recorded on Bruker AV 400 MHz, AV-III 400 MHz
and AV-500 MHz instruments and were calibrated using a residual undeuterated solvent as an
internal reference (d-chloroform, 1H NMR δ 7.26 ppm, 13C NMR δ 77.1 ppm; d4-methanol, 1H
NMR δ 3.31 ppm, 13C NMR δ 49.0 ppm). The abbreviations were used to interpret NMR peak
multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad.
HR MALDI-mass spectra were conducted on an Applied Biosystems 4800 Proteomics
Analyzer equipped with an Nd/YAG laser (335 mm) operating at a repetition rate of 200 Hz.
HR FAB and HR EI-mass spectra were conducted on a JEOL JMS-700 double-focusing mass
spectrometer with a resolution of 8000 (5% valley definition). HR ESI-mass spectra were
conducted on a dual ionization ESCi® (ESI/APCi) source options, Waters LCT premier XE
(Waters Corp., Manchester, UK). Optical rotations were obtained on a Jasco P-2000
polarimeter (1-dm cell). Infrared (IR) spectra were recorded on a Thermo Nicolet iS-5 FT-IR
spectrometer. Melting points were recorded on a BÜCHI M-565 melting point apparatus.
[(9Z,12Z)-N-((3R,4R,5S)-4,5-Dimethyl-2-oxotetrahydrofuran-3-yl)
octadeca-9,12-dienamide] (N55):
Data of N55 from isolated sample purified by high-performance liquid chromatography
11

21
(HPLC): Rf = 0.28 [ethyl acetate (EtOAc)/hexane = 1/2, I2]; [α]D = –23.0 (c 0.30, CHCl3,
5-cm cell); IR (film) ν = 3303, 3008, 2956, 2926, 2854, 1782, 1657, 1650, 1536, 1461, 1453,
1388, 1183, 1047, 908, 723 cm-1; 1H NMR (500 MHz, CD3OD) δ 5.39–5.29 (m, 4 H), 4.38 (d,
J =11.8 Hz, 1 H), 4.19 (dt, J = 9.8, 6.1 Hz, 1H), 2.78 (t, J = 6.5 Hz, 2 H), 2.26 (t, J = 7.4 Hz, 2
H), 2.17–2.04 (m, 5 H), 1.66–1.60 (m, 2 H), 1.41 (d, J = 6.1 Hz, 3 H), 1.40-1.28 (m, 14 H),
1.13 (d, J = 6.6 Hz, 3H), 0.91 (t, J = 6.8 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ 176.5,
176.2, 130.9, 130.9, 129.1, 129.1, 81.4, 57.5, 45.6, 36.9, 32.7, 30.7, 30.5, 30.3, 30.3, 30.2,
28.2, 26.8, 26.5, 23.6, 18.8, 14.4, 14.0; HRMS (ESI-TOF) m/z [M + H]+ calcd. for C24H42O3N
392.3165, found 392.3160.
21
Data of synthetic N55: Rf = 0.28 (EtOAc/hexane = 1/2, I2); [α]D = –21.8 (c 0.30, CHCl3,
5-cm cell); IR (film) ν = 3303, 3009, 2957, 2927, 2855, 1782, 1657, 1650, 1533, 1461, 1454,
1389, 1187, 1047, 908, 723 cm-1; 1H NMR (500 MHz, CD3OD) δ 5.39–5.29 (m, 4 H), 4.38 (d,
J =11.8 Hz, 1 H), 4.19 (dt, J = 9.8, 6.1 Hz, 1H), 2.78 (t, J = 6.5 Hz, 2 H), 2.26 (t, J = 7.5 Hz, 2
H), 2.17–2.04 (m, 5 H), 1.66–1.60 (m, 2 H), 1.41 (d, J = 6.1 Hz, 3 H), 1.40-1.28 (m, 14 H),
1.13 (d, J = 6.6 Hz, 3H), 0.91 (t, J = 6.9 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ 176.5,
176.2, 130.9, 130.9, 129.1, 129.1, 81.4, 57.5, 45.6, 36.9, 32.7, 30.7, 30.5, 30.3, 30.3, 30.2,
28.2, 26.8, 26.5, 23.6, 18.8, 14.4, 14.0; HRMS (MALDI-TOF) m/z [M + Na]+ calcd. for
C24H41O3NNa 414.2979, found 414.2963.
Ethyl (2R,3R)-2-(4-Methoxyphenylamino)-3-methyl-4-oxopentanoate (4):1,2 Na2SO4 (10.7
g, 75.0 mmol) was added to a stirred solution of 4-anisidine (2) (3.69 g, 30.0 mmol) in
toluene (PhMe) (30.0 mL), followed by the addition of ethyl glyoxalate (1) (6.13 mL, 30.0
mmol, 50% in toluene) within 10 to 20 min. The reaction mixture was stirred at room
temperature for 30 min. After the starting material was consumed, Na2SO4 was filtered out by
Celite, and the filtrate was concentrated under reduced pressure to give a brown oil (3) that
was used immediately for the next step without further purification. A solution of 3 in dry
dimethylformamide (DMF) (15.5 mL) was slowly added to a stirred solution of butanone
(59.0 mL, 660 mmol) and D-proline (1.21 g, 10.5 mmol) in dry DMF (46.6 mL) over 30 min
12

at room temperature, and the resulting mixture was stirred at room temperature for 12 h. After
the starting material was consumed, the reaction mixture was filtered through a pad of sieve
and concentrated under reduced pressure. The resulting yellow oil containing 4 and its
regioisomer (9; 5 : 1) was directly used for the next reaction without further purification. A
small amount of mixture was applied to column chromatography (silica gel, EtOAc/hexane =
1/4) and then HPLC for the characterization of 4. Rf = 0.25 (EtOAc/hexane = 1/5, UV);
23.1
[α]D = 47.3 (c 1.05, CHCl3, >99% ee); IR (film) ν = 3379, 2981, 2936, 1729, 1713, 1514,
1
1235, 1200, 1180, 1035, 823 cm-1; H NMR (400 MHz, CDCl3) δ 6.77 (d, J = 8.8 Hz, 2 H),
6.66 (d, J = 8.8 Hz, 2 H), 4.31 (d, J = 5.8 Hz, 1 H), 4.19–4.13 (m, 2 H), 3.74 (s, 3 H), 3.03 (m,
1 H), 2.23 (s, 3 H), 1.25 (d, J = 7.1 Hz, 3 H), 1.22 (t, J = 7.2, 3 H); 13C NMR (125 MHz,
CDCl3) δ 209.3, 172.9, 153.3, 140.9, 116.0, 115.0, 61.5, 59.8, 55.8, 49.4, 28.6, 14.3, 12.4;
enantioselectivity was determined by HPLC analysis (Chiralpak-AS, 1.0 mL/min, 220 nm,
hexane/i-PrOH 97/3); retention times were 21.7 (enantiomer) and 30.9 min (major).
Ethyl (2R,3R)-2-Amino-3-methyl-4-oxopentanoate (5):3,4 A solution of (NH4)2S2O8 (913
mg, 4.0 mmol) and cerium ammonium nitrate (CAN) (110 mg, 0.2 mmol) in H2O (5.8 mL)
was slowly added to a stirred solution of the mixture of 4 and its regeoisomer (9; 5 : 1, 547
mg, ~2.0 mmol) in acetonitrile (MeCN) (1.0 mL) at 0 °C. The resulting mixture was then
heated to 35 °C and stirred for 3 h. Upon the completion of the reaction, the reaction mixture
was diluted with H2O (5.8 mL) and washed with CH2Cl2 (5 mL × 4). The aqueous layer was
basified with 1 M Na2CO3 aqueous solution to pH ~ 8 and then extracted with CH2Cl2 (15 mL
× 5). The combined organic layers were dried over Na2SO4 and concentrated under reduced
pressure to give a brown liquid that contains 5 and its regioisomer (10, 229 mg). The crude
mixture was used immediately for the next reaction without further purification. Data of 5
1
from pure 4: Rf = 0.40 (methanol/CH2Cl2 = 1/10, ninhydrin); H NMR (400 MHz, CDCl3) δ
4.19–4.13 (m, 2 H), 3.86 (d , J = 4.8 Hz, 1 H), 2.91 (m, 1 H), 2.19 (s, 3 H), 1.25 (t, J = 7.1 Hz,
3 H), 1.11 (d, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 209.9, 174.3, 61.3, 55.4, 49.8,
28.4, 14.2, 11.0; HRMS (APCI-TOF) m/z [M + H]+ calcd. for C8H16O3N 174.1130, found
174.1127.
13

Ethyl (2R,3R)-2-(Dibenzylamino)-3-methyl-4-oxopentanoate (6): A suspension of the
crude 5, its regeoisomer (10, 5 : 1, 98 mg, ~0.57 mmol) and K2CO3 (235 mg, 1.7 mmol) in
DMF/H2O (1.1 mL/0.11 mL) was stirred at room temperature for 15 min, followed by the
dropwise addition of benzyl bromide (BnBr) (0.34 mL, 2.8 mmol). After stirring at 60 °C for
18 h, H2O (5 mL) was added, and the reaction mixture was then extracted with EtOAc (10 mL
× 3). The combined organic layers were dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified by flash column chromatography on silica gel
(EtOAc/hexane = 1/15) to give 6 as a white solid (120 mg, 0.34 mmol, 60%). Rf = 0.45
23
(EtOAc/hexane = 1/5, UV); mp 58.8–62.3 °C; [α]D = 186 (c 1.03, CHCl3); IR (film) ν =
3062, 3029, 2977, 2929, 2852, 1723, 1602, 1495, 1454, 1370, 1201, 1169, 1026, 961, 748,
699 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.37–7.22 (m, 10 H), 4.34–4.16 (m, 2 H), 3.86 (d, J
= 13.5 Hz, 2 H), 3.49 (d, J = 10.9 Hz, 1 H), 3.45 (d, J = 13.4 Hz, 2 H), 3.08 (dt, J = 10.9, 7.2
Hz, 1 H), 2.15 (s, 3 H), 1.38 (t, J = 7.1 Hz, 3 H), 1.10 (d, J = 7.3 Hz, 3 H); 13C NMR (100
MHz, CDCl3) δ 211.5, 171.9, 138.9, 129.2, 128.4, 127.3, 62.6, 60.5, 55.2, 46.0, 29.2, 14.7,
14.5; HRMS (ESI-TOF) m/z [M + H]+ calcd. for C22H28O3N 354.2069, found 354.2061.
(3R,4R,5S)-3-(Dibenzylamino)-4,5-dimethyldihydrofuran-2(3H)-one (7): L-selectride
[0.54 mL, 1.0 M in tetrahydrofuran (THF), 0.54 mmol] was added to a stirred solution of 6
(173 mg, 0.49 mmol) in dry THF (4.9 mL) at –78 °C under nitrogen. After stirring for 2 h
at –78 °C, the reaction mixture was poured into a vigorously stirred mixture of EtOAc/1 M
HCl aqueous solution (10.0 mL/10.0 mL). The aqueous layer was extracted with EtOAc (10
mL × 3). The combined organic layers were washed with H2O (5 mL × 3–5) until pH ~ 6 and
then washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel (EtOAc/hexane = 1/20,
UV) to give 7 as a white solid (143 mg, 0.46 mmol, 94%). Rf = 0.33 (EtOAc/hexane = 1/10,
21
UV); mp 66.5–69.7 °C; [α]D = 89.8 (c 1.06, CHCl3); IR (film) ν = 3062, 3028, 2972, 2928,
2850, 1769, 1601, 1493, 1454, 1385, 1325, 1236, 1185, 1171, 1141, 1050, 995, 953, 745, 699
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.42–7.40 (m, 4 H), 7.35–7.31 (m, 4 H), 7.28–7.23 (m, 2
H), 4.00 (d, J = 13.8 Hz, 2 H), 3.91–3.83 (m, 3 H), 3.29 (d, J = 11.8 Hz, 1 H), 2.05 (m, 1 H),
14

1.35 (d, J = 6.1 Hz, 3 H), 1.02 (d, J = 6.5 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 175.3,
139.4, 128.8, 128.4, 127.3, 79.6, 65.4, 54.9, 42.1, 18.9, 14.1; HRMS (ESI-TOF) m/z [M +
Na]+ calcd. for C20H23O2NNa 332.1626, found 332.1623.
(3R,4R,5S)-3-Amino-4,5-dimethyldihydrofuran-2(3H)-one (8):5,6 Pd(OH)2/C (6.2 mg, 20%)
was added to a stirred solution of 7 (61.9 mg, 0.20 mmol) in EtOAc (4.0 mL) under nitrogen
and then purged with hydrogen (1 atm, balloon) for an hour at room temperature. After
stirring at room temperature under hydrogen for 18 h, the reaction mixture was filtered
through Celite and concentrated under reduced pressure to give 8 as a colorless oil (25.8 mg,
0.20 mmol, >99%) without further purification. Rf = 0.4 (methanol/CH2Cl2 = 1/10, ninhydrin);
[α]D24 = 29.0 (c 0.98, CHCl3); IR (film) ν = 3374, 3310, 2973, 2927, 2878, 2852, 1771, 1456,
1
1389, 1330, 1192, 1145, 1044, 983, 946, 918, 735, 702 cm-1; H NMR (400 MHz, CDCl3) δ
4.05 (dq, J = 9.9, 6.1 Hz, 1 H), 3.25 (d, J = 5.2 Hz, 1 H), 1.80 (m, 1 H), 1.73 (s, 2 H), 1.42 (d,
J = 6.2 Hz, 3 H), 1.21 (d, J = 6.6 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 178.2, 79.8, 58.9,
47.5, 18.6, 14.2; HRMS (EI+) m/z M+ calcd. for C6H11O2N 129.0790, found 129.0791.
(9Z,12Z)-N-((3R,4R,5S)-4,5-Dimethyl-2-oxotetrahydrofuran-3-yl)octadeca-9,12-dienami
de (N55): Benzotriazol-1-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (62.5
mg, 0.12 mmol) was added to a stirred solution of 8 (12.9 mg, 0.10 mmol) and linoleic acid
(31 μL, 0.10 mmol) in dry DMF (1.0 mL), followed by freshly distilled
N,N-diisopropylethylamine (DIPEA) (21 μL, 0.12 mmol) at room temperature under nitrogen.
The reaction mixture was stirred for 18 h. After the starting material was consumed, the
reaction mixture was diluted with EtOAc (10 mL) and washed with H2O (5 mL) and brine (5
mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel (EtOAc/hexane = 1/4) to
give N55 as a colorless oil (36.9 mg, 0.094 mmol, 94%). Rf = 0.28 (EtOAc/hexane = 1/2, I2);
[α]D21 = –21.8 (c 0.30, CHCl3, 5-cm cell); IR (film) ν = 3303, 3009, 2957, 2927, 2855, 1782,
15

1657, 1650, 1533, 1461, 1454, 1389, 1187, 1047, 908, 723 cm-1; 1H NMR (500 MHz, CD3OD)
δ 5.39–5.29 (m, 4 H), 4.38 (d, J =11.8 Hz, 1 H), 4.19 (dt, J = 9.8, 6.1 Hz, 1H), 2.78 (t, J = 6.5
Hz, 2 H), 2.26 (t, J = 7.5 Hz, 2 H), 2.17–2.04 (m, 5 H), 1.66–1.60 (m, 2 H), 1.41 (d, J = 6.1
Hz, 3 H), 1.40-1.28 (m, 14 H), 1.13 (d, J = 6.6 Hz, 3H), 0.91 (t, J = 6.9 Hz, 3H); 13C NMR
(125 MHz, CD3OD) δ 176.5, 176.2, 130.9, 130.9, 129.1, 129.1, 81.4, 57.5, 45.6, 36.9, 32.7,
30.7, 30.5, 30.3, 30.3, 30.2, 28.2, 26.8, 26.5, 23.6, 18.8, 14.4, 14.0; HRMS (MALDI-TOF)
m/z [M + Na]+ calcd. for C24H41O3NNa 414.2979, found 414.2963.
References
1. Córdova, A., Notz, W., Zhong, G., Betancort, J. M., Barbas III, C. F. (2002) A
highly enantioselective amino acid-catalyzed route to functionalized γ-amino acids. J. Am.
Chem. Soc. 124 1842–1843
2. Marin, S. D. L., Catala, C., Kumar, S. R., Valleix, A., Wagner, A., Mioskowski, C.
(2010) A practical and efficient total synthesis of potent insulinotropic
(2S,3R,4S)-4-hydroxyisoleucine through a chiral N-protected γ-keto-α-aminoester. Eur. J.
Org. Chem. 3985–3989
3. Mioskowski, C., Marin, S. D. L., Maruani, M., Gill, M. (2006) Analogs of
4-hydroxyisoleucine and uses thereof. US Patent 20060199853 (A1)
4. Chapal, N., McNicol, P., Jette, L. (2006) Compounds and compositions for use in the
prevention and treatment of obesity and related syndromes. US Patent 20060223884 (A1)
5. Tamura, O., Iyama, N., Ishibashi, H. (2004) Syntheses of (–)-funebrine and (–)-funebral,
using sequential transesterification and intramolecular cycloaddition of a chiral nitrone. J.
Org. Chem. 69, 1475–1480
6. Yuen, T.-Y., Eaton, S. E., Woods, T. M., Furkert, D. P., Choi, K. W., Brimble, M. A.
(2014) A Maillard approach to 2-formylpyrroles: synthesis of magnolamide, lobechine
and funebral. Eur. J. Org. Chem. 1431–1437
16

HPLC analysis
HPLC condition: Chiralpak-AS, 1.0 mL/min, 220 nm, hexane/i-PrOH 97/3, 21.7 (minor) and
30.9 min (major).
Racemic 4:
4 with >99% ee:
17

Figure S1. The MS/MS spectra of N55
18

1
Table S1. Comparative H and 13C NMR spectra data between isolated N55 and synthetic
N55
Source Isolated N55 a
Synthetic N55 a
Isolated N55 a
δC (ppm)
176.2 (s)
57.5 (d)
Synthetic N55 a
δC (ppm)
176.2 (s)
57.5 (d)
Position
δ
H (ppm)
δ
H (ppm)
-
-
2
3
4.38 (d, J = 11.8 Hz)
2.17-2.04 (m)
4,38 (d, J = 11.8 Hz)
2.17-2.04 (m)
4
45.6 (d)
45.6 (d)
5
4.19 (dt, J = 9.8, 6.1 Hz)
1.13 (d, J = 6.6 Hz)
1.41 (d, J = 6.1 Hz)
-
4.19 (dt, J = 9.8, 6.1 Hz)
1.13 (d, J = 6.6 Hz)
1.41 (d, J = 6.1 Hz)
-
81.4 (d)
81.4 (d)
6
14.0 (q)
14.0 (q)
7
18.8 (q)
18.8 (q)
1’
176.5 (s)
36.9 (t)
176.5 (s)
36.9 (t)
2’
2.26 (t, J = 7.4 Hz)
1.66-1.60 (m)
2.26 (t, J = 7.5 Hz)
1.66-1.60 (m)
3’
26.8 (t)
26.8 (t)
4’-7’ & 15’-17’
8’ & 14’
1.40-1.28 (m)
1.40-1.28 (m)
33-23 (t)
28.2 (t)
33-23 (t)
28.2 (t)
2.06 (m)
2.06 (m)
9’, 10’, 12’ & 13’
11’
5.39-5.29 (m)
5.39-5.29 (m)
130.9 & 129.1 (d)
26.5 (t)
130.9 & 129.1 (d)
26.5 (t)
2.78 (t, J = 6.5 Hz)
0.91 (t, J = 6.8 Hz)
2.78 (t, J = 6.5 Hz)
0.91 (t, J = 6.9 Hz)
18’
14.4 (q)
14.4 (q)
a 500 MHz NMR in d4-methanol (δH = 3.31 ppm; δC = 49.0 ppm)
19

1
Figure S2. Comparative 500 MHz H NMR spectra between isolated N55 (upper) and
synthetic N55 (lower) in d4-methanol (δH = 3.31 ppm)
Figure S3. Comparative 500 MHz 13C NMR spectra between isolated N55 (upper) and
synthetic N55 (lower) in d4-methanol (δC = 49.0 ppm)
20

Signal Transduction:
Isolation of Positive Modulator of
Glucagon-like Peptide-1 Signaling from
Trigonella foenum-graecum (Fenugreek)
Seed
Klim King, Nai-Pin Lin, Yu-Hong Cheng,
Gao-Hui Chen and Rong-Jie Chein
J. Biol. Chem. 2015, 290:26235-26248.
doi: 10.1074/jbc.M115.672097 originally published online September 2, 2015
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