Antidiabetic in vitro and in vivo evaluation of cyclodipeptides isolated from Pseudomonas fluorescens IB-MR-66e

Article abstract of DOI:10.1039/c9nj00645a

Three cyclodipeptides [cyclo(l-Pro-l-Leu), 1; cyclo(l-Pro-l-Val), 2; and cyclo(l-Pro-l-Phe), 3] were isolated from Pseudomonas fluorescens IB-MR-66e. The structures were established by spectral means and corroborated by synthesis. The antidiabetic potential of compounds 1-3 was explored in vivo, in vitro and in silico. The three peptides showed important inhibitory activity against the α-glucosidase enzyme. Further analysis in vivo using a sucrose tolerance test corroborated that compounds 1 and 3 (1-30 mg kg^-1) significantly reduced the postprandial state. Peptide 1 (1-30 mg kg^-1) also reduced the postprandial peak after a glucose challenge and exhibited significant hypoglycemia during an insulin tolerance test; thus, its antidiabetic action involved also an improvement of insulin utilization not related to Akt phosphorylation nor to an increment in mitochondrial bioenergetics nor insulin secretion.

Full text of DOI:10.1039/c9nj00645a

NJC
View Article Online
View Journal
PAPER
Antidiabetic in vitro and in vivo evaluation of
cyclodipeptides isolated from Pseudomonas
fluorescens IB-MR-66e†
Cite this:DOI: 10.1039/c9nj00645a
a
a
a
M. Lozano-Gonzalez,
B. Ovalle-Magallanes, M. Rangel-Grimaldo,
´
b
c
d
c
S. De la Torre-Zavala, L. G. Noriega, C. Tovar-Palacio, A. R. Tovar and
a
R. Mata
*
Three cyclodipeptides [cyclo(L-Pro-L-Leu), 1; cyclo(L-Pro-L-Val), 2; and cyclo(L-Pro-L-Phe), 3] were
isolated from Pseudomonas fluorescens IB-MR-66e. The structures were established by spectral means
and corroborated by synthesis. The antidiabetic potential of compounds 1–3 was explored in vivo,
in vitro and in silico. The three peptides showed important inhibitory activity against the a-glucosidase
enzyme. Further analysis in vivo using a sucrose tolerance test corroborated that compounds 1 and 3
Received 8th February 2019,
Accepted 29th April 2019
À1
À1
(
1–30 mg kg ) significantly reduced the postprandial state. Peptide 1 (1–30 mg kg ) also reduced the
postprandial peak after a glucose challenge and exhibited significant hypoglycemia during an insulin
tolerance test; thus, its antidiabetic action involved also an improvement of insulin utilization not related
to Akt phosphorylation nor to an increment in mitochondrial bioenergetics nor insulin secretion.
DOI: 10.1039/c9nj00645a
rsc.li/njc
2
,4-diacetylphloroglucinols, pseudomonic acid A derivatives,
1
Introduction
3–6
7
3,8
phenazines, pyrrole derivatives, indole alkaloids, pyochelin
9
Type 2 diabetes (T2DM) is a metabolic disease characterized by type peptides, and cyclodipeptides (CDPs) of the diketopiperazine
insulin deficiency resulting from inadequate insulin secretion (DKP) family. Among the last group of compounds P. fluorescens
or resistance. The global prevalence of T2DM is rapidly increasing; biosynthesizes cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Tyr), cyclo(L-Pro-
10
1
thus, according to the International Diabetes Federation, in L-Phe) and cyclo(L-Pro-L-Leu). The first two are toxic to cells and
1
1
2
017 there were 425 million people with this disease worldwide, seedlings of Pinus thunbergii, while the other two are effective
2
6
and 1% of these patients died from associated complications.
Therefore, there is an urgent need to discover new therapeutic
alternatives to treat this important health problem. In this and roasted food.
antifungal agents.
The CDPs of the DKP family are also found in fungi, chocolate
It has been suggested that their presence in
scenario, herein we have explored the potential of Pseudomonas food is the result of degradation of larger peptides.
12
13,14
12–14
In micro-
fluorescens Migula (Pseudomonadaceae), isolated from mineral organisms these compounds are assembled by the catalysis of
15
soils in Fresnillo, Zacatecas, Mexico, as a source of antidiabetic CDP-synthases.
compounds. DKPs have recently gained interest due to their wide array of
P. fluorescens is a Gram-negative rod-shaped bacterium biological activities, including antibacterial, anti-fungal, phyto-
16
17
18,19
20
21
found in a wide range of environments, including the rhizosphere, toxic, antiviral,
anti-prion and antitumor. Furthermore, a
mammalian hosts, and soil, as well as plants and indoor wall recent Japanese patent claims that some DKPs are a-glucosidase
3
22
surfaces, just to mention the most important. P. fluorescens inhibitors (AGIs). However, no in vivo test has supported this
biosynthesizes a few important secondary metabolites comprising finding. AGIs are important therapeutic agents for treating the
prediabetic condition and modulating post-prandial hypergly-
2
3
a
b
c
caemia in combination with first line antidiabetic agents.
Facultad de Qu ´ı mica, Departamento de Farmacia, Universidad Nacional
Aut o´ noma de M ´e xico, Ciudad de M ´e xico, Mexico. E-mail: rachel@unam.mx
Instituto de Biotecnolog ´ı a, Universidad Aut o´ noma de Nuevo Le o´ n, Monterrey,
Mexico
As a part of a bioprospection program for the search of new
antidiabetic agents, herein we describe the activity of a few
dipeptides from P. fluorescens on yeast a-glucosidase using a
well-known enzymatic assay. Furthermore, different in vivo and
cell-based evaluations were carried out in order to assess the
CDPs’ effects on glycaemia, insulin secretion and mitochondrial
Instituto Nacional de Ciencias M ´e dicas y Nutrici o´ n ‘‘Salvador Zubir a´ n’’,
Departamento de Fisiolog ´ı a de la Nutrici o´ n, Ciudad de M ´e xico, Mexico
Instituto Nacional de Ciencias M ´e dicas y Nutrici o´ n ‘‘Salvador Zubir a´ n’’,
Departamento de Nefrolog ´ı a y Metabolismo Mineral, Ciudad de M ´e xico, Mexico
d
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nj00645a respiration.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
New J. Chem.

View Article Online
Paper
NJC
2
Materials and methods
materials (triplicated) were incubated for 10 min with 20 mL of
enzyme stock solution (0.4 units mL in phosphate buffer
solution 100 mM, pH 7). After incubation, 10 mL of substrate
À1
2.1 General experimental procedures
Melting points are uncorrected and were determined on a Fisher-
Johns apparatus. IR spectra were recorded using a PerkinElmer
(
pNPG 5 mM) was added and further incubated for 30 min at
3
7 1C, and the absorbance of each sample was determined. The
4
00 FT-IR spectrophotometer (Waltham, MA, USA). NMR spectra,
including bidimensional, were recorded in CD OD or CDCl
solutions on a Bruker-Avance III HD spectrometer (Billerica, 2.5 Theory calculation
25
inhibitory activity was expressed as IC50 as previously reported.
3
3
1
13
MA, USA) at either 500 MHz ( H) or 125 MHz ( C) using
tetramethylsilane as an internal standard.
Column chromatography (CC) was carried out using silica
gel. Flash chromatography was carried out with a Teledyne
The minimized structures for docking simulations were prepared
using Autodock Tools package v1.5.4 (ADT, http://mgltools.scripps.
25
edu/) as previously described.
CombiFlashRf + Lumen (Thousand Oaks, CA, USA) chromatograph 2.6 Animals
equipped with a photodiode array detector and an evaporative light
Experimental procedures were implemented in accordance
scattering detector, using a RediSepRf high-performance GOLD
silica gel column and eluting with a gradient of hexane, chloro-
form, and methanol. Thin layer chromatographic analyses were
performed on silica gel 60 F254 plates (Merck, Kenilworth, NJ,
USA), and visualization of the plates was carried out using a
Ce (SO ) (10%) solution in H SO .
with internationally accepted principles for laboratory animal
use and care and following the Mexican Official Norm for
Laboratory Animal Care and Use (NOM-062-ZOO-1999). The
Institutional Committee for Care and Use of Laboratory Animals
(
CICUAL-FQ) of Facultad de Qu ´ı mica UNAM approved the proto-
2
4 3
2
4
cols (FQ/CICUAL/232-2/17 and FQ/CICUAL/334/18). Evaluations
were conducted on 4-week old male ICR mice, obtained from
Centro UNAM-Envigo (Envigo RMS S.A. de C.V.). Animals were
held under a controlled temperature (22 Æ 2 1C) on a light–dark
2.2 Bacterial material
Pseudomonas fluorescens strain IB-MR-66 was isolated from
mineral soils randomly collected in Fresnillo Zacatecas, Mexico,
(12 h–12 h) cycle, with free access to a standard diet and purified
2
4
in August 2014, as previously described. The bacterium was
identified based on morphological characteristics and sequence
data of the [16S ribosomal RNA] region was deposited in
GenBank as accession MH810270. Data available at GenBank
aligning with strain IB-MR-66 suggested this bacterium is
P. fluorescens.
water before experiments. Six-to-eight normoglycaemic and/or
hyperglycaemic mice were grouped for each experiment. Blood
samples were obtained by venipuncture on the lateral saphenous
vein of the right leg using a 31 G Â 5 mm needle. Glycaemia
À1
(
mg dL ) was measured using a commercial glucometer (One
Touch Ultra 2, Johnson & Johnson, NJ, USA). At the end of the
26
experiments, mice were euthanized by hypoxia in a CO
2
chamber.
2.3 Fermentation, extraction, and isolation
For organ and tissue recovery, mice were euthanized by porta-
A single colony of P. fluorescens was inoculated into Luria Broth (LB) exsanguination after isoflurane anaesthesia.
medium at 25 1C for 2 days. Then, the culture was inoculated into
2.7 Sample preparation and route of administration
1.5 litres LB medium in a 2 L flask and cultured under agitation at
2
5 1C for 7 days. A 1 : 1 mixture of MeOH–CH Cl was added to the Three logarithmic doses of CDPs 1–3 and single doses of positive
2
2
final culture. A liquid–liquid extraction was carried out (300 mL Â 3) controls acarbose and metformin were suspended in a vehicle
and the organic phase was evaporated to dryness under a vacuum. consisting of isotonic saline solution with 0.05% Tween 80 and
The extract was suspended in 150 mL of a mixture of MeCN–MeOH given per os (p.o., orally) using a gavage feeding needle (18 G, 35 mm;
s
(
1 : 1) and partitioned with n-hexane (8 Â 150 mL). The combined 0.2 mL per 10 g bodyweight). Insulin (Humulin , Indianapolis IN,
MeCN–MeOH fractions were dried under a vacuum, and the USA) was dissolved in sterile isotonic saline solution and intra-
resulting residue (0.3 g) was dissolved in CH Cl and subjected peritoneally administered (0.1 mL per 10 g bodyweight).
to flash chromatography using a gradient of n-hexane–CHCl3
0.0–10.0 min, 100 : 0 - 0 : 100) and CHCl –MeOH (10.0–17.5 min,
2
2
2
.8 Oral carbohydrate tolerance tests
00 : 0, 17.5–25.0 min, 100 : 0 - 98 : 2; 25.0–32.5 min, 98 : 2 - 95 : 5; Normoglycaemic mice were deprived of food 4 h before the
2.5–45.0 min, 95 : 5 - 90 : 10; 45.0–65.0 min, 90 : 10 - 80 : 20, experiment with free access to drinking water. After fasting,
(
1
3
3
6
5.0–90.0 min, 80 : 20 - 0 : 100). The separation process yielded basal glycaemia was registered. For the sucrose tolerance test,
À1
nine primary fractions (F1–F9). Based on their yield F7 and F9 were the mice received the vehicle, or acarbose (5 mg kg p.o.), or
further purified. F7 (45 mg) was subjected to open silica gel column compounds 1 and 3 (1, 10 and 30 mg kg p.o.). Half-an-hour
eluting with CH
À1
À1
2
Cl
2
–MeOH (80 : 20) to yield 1 (5 mg). F9 (35 mg), later, all animals received an oral sucrose challenge (2 g kg ).
was also purified by silica column chromatography eluting with Blood samples were collected at 10, 20, 30, 60, 90 and 120 min
CH Cl –MeOH (70 : 30) to yield 3 (3 mg).
after carbohydrate load. Oral glucose tolerance tests for CDPs 1
and 3 were conducted using a similar experimental design in
2
2
2.4 Inhibition of a-glucosidases
both normoglycaemic and hyperglycaemic mice, but using
À1
The bacterial extract, fractions, compounds, and acarbose (positive metformin (200 mg kg ) as a positive control, and a glucose
control) were dissolved in MeOH. Aliquots of 0–40 mL of testing challenge of 1 g kg p.o.
À1
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019

View Article Online
NJC
Paper
2
.9 Insulin tolerance test and organ recovery
concentration using an ELISA kit. The total insulin content was
determined in an aliquot obtained by addition of acidified ethanol
Normoglycaemic mice were deprived of food for 4 h before the
experiment. After this time, basal glycaemia was measured,
and the mice received either the vehicle, or a single dose of
(75% ethanol/1.5% HCl) to the attached cells. The insulin secretion
was expressed as the percentage of insulin secreted into the media
with respect to the sum of the secreted and total insulin content.
À1
À1
compound 1 (30 mg kg ) or 3 (10 mg kg ). Thirty minutes
after treatment, the mice received an intraperitoneal dose of
2
.12 Mitochondrial respiration
À1
0
3
.05 U kg of insulin. Glycaemia was measured at 10, 20 and
0 minutes following insulin administration. In an independent
A Seahorse Extracellular Flux (XF) 96 Analyzer (Seahorse Bioscience,
Inc, North Billerica, MA, USA) was used to measure the oxygen
consumption rate (OCR) in mouse myoblast cells C2C12 after
sequential addition of oligomycin (2 mM), FCCP (0.5 mM) and a
mixture of rotenone plus antimycin A (0.5 mM) to the respiring cells.
Basal mitochondrial and ATP-linked respiration, proton leak and
but similar experimental design, liver, adipose tissue and gas-
trocnemius muscle were obtained from vehicle, insulin and
compound 1 + insulin treated mice (n = 3). The organs were
removed after 20 min of the administration of insulin. Samples
were kept in liquid nitrogen and stored at À70 1C until use.
27
the spare capacity were then calculated as previously described.
The OCR was measured after the cells were incubated for 18 h with
compound 1 (10 mg mL ).
2
.10 Western blot analysis
À1
Frozen liver, adipose tissue and gastrocnemius muscle were
homogenized in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 2.13 Data treatment and statistical analyses
1
1
mM EDTA, 5 mM Na P O , 1 mM Na VO , 50 mM NaF and
4 2 7 3 4
% NP-40) with protease inhibitor cocktail (cOmplete, Roche,
Glycaemia values were used to calculate the percentage of glycaemia
variation. Furthermore, areas under the curve (AUC) applying the
26
Merck, Mexico). The protein concentration was determined
using the DC protein assay kit (Bio-Rad Laboratories, Richmond,
CA). Tissue lysates (20 mg) were combined with Laemmli sample
buffer and separated by SDS-PAGE. After electrophoretic separation,
the proteins were electrotransferred to a PVDF membrane using a
Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules,
CA; USA), blocked and incubated overnight at 4 1C with the anti-AKT
trapezoidal rule were obtained. The results are expressed as the
mean Æ SEM of glycaemia variation or AUC. Statistical significance
(
(
p o 0.05) was assessed with the GraphPad Prism software
version 5.0; GraphPad Inc., La Jolla, CA, USA) using one-way
or two-way ANOVA tests followed by an appropriate post hoc test.
3
Results and discussion
(
sc-8312, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-
AKT (9018, Cell Signaling, Technology, Inc., Danvers, MA, USA) and
anti-actin antibody (sc-1615, Santa Cruz Biotechnology, Dallas, TX,
USA). Band detection was carried out using a chemiluminescent
Western blotting kit (Immobilon Western Chemiluminescent HRP
Substrate, Millipore, Burlington, MA, USA). Digital images of the
membranes were obtained using a ChemiDoc MP densitometer and
processed by Image Lab software (Bio-Rad, Hercules, CA, USA).
Results are reported relative to Akt and b-actin. Blots were quanti-
fied using NIH Image J software (v. 1.49, National Institutes
of Health, USA).
3.1 Isolation, synthesis and characterization
An organic extract from the culture of P. fluorescens inhibited
the activity of yeast a-glucosidase in a concentration dependent
manner (IC50 = 1000 ppm). Fractionation of the extract led to
the isolation of the three CPDs (1–3) shown in Fig. 1.
The compounds were characterized by spectral and synthetic
means as cyclo(L-Pro-L-Leu) (1), cyclo(L-Pro-L-Val) (2), and cyclo-
2
8–30
(L-Pro-L-Phe) (3).
The synthesis of 1–3 was performed as
2
8
previously described (Scheme 1), but using monowave Anton
2.11 INS-1E cell culture and insulin secretion assays
Rat insulinoma INS-1E cells (provided by Dr P Maechler, University
of Geneva, Switzerland) were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum, 1 mM sodium pyruvate,
5
1
0 mM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES,
À1
À1
00 U mL
penicillin, and 100 mg mL streptomycin. For
insulin secretion assays, rat insulinoma INS-1E cells were seeded
À1
on 24-well plates and after 48 h treated with 0, 5, 10 or 20 mg mL
of compound 1 for 2 h. After treatment, rat insulinoma INS-1E cells
were washed 3 times with Krebs-Ringer bicarbonate buffer (KRBB:
1
35 mM NaCl, 3.6 mM KCl, 2 mM NaHCO
3 2 4
, 0.5 mM NaH PO ,
0
.5 mM MgSO , 1.5 mM CaCl , 10 mM HEPES, pH 7.4, and 0.1%
4
2
bovine serum albumin) containing 2.5 mM glucose. Rat insulinoma
INS-1E cells were incubated for 30 min in KRBB containing 2.5 mM
glucose and then maintained on 2.5 mM glucose or switched
to 8.3 mM glucose for an additional 1 h. After incubation, the
medium was collected and frozen in order to determine the insulin Fig. 1 CPDs isolated from the culture of P. fluorescens.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
New J. Chem.

View Article Online
Paper
NJC
Scheme 1 General synthesis of dipeptides 1–3.
Fig. 3 Model of binding for compound 1 and acarbose (purple and
orange sticks respectively) with aGHY (green cartoon, PDB 3A4A).
Paar equipment for heating at 120 1C for 1 h 30 min. This heating
method allowed faster results than that of an autoclave at 180 1C
for 24 h previously used.
28
À1
only at a dose of 30 mg kg (Fig. 2, panel B). Compound 2 was
inactive. The in vivo effects of 1 and 3 are consistent with the
inhibitory action of CPDs on a-glucosidases.
3.2 Inhibition of a-glucosidases
Compounds 1–3 were tested for their ability to inhibit yeast
a-glucosidase activity. The IC s calculated were 4.6 Æ 0.2, 3.2 Æ
3
.4 Docking
5
0
0
.5 mM and 3.4 Æ 0.6, respectively versus 1.6 Æ 0.1 mM for In order to envisage the putative binding mode of the most
acarbose. Interestingly when the synthetic lineal peptides (1a–3a) active compound 1, a docking analysis was carried out using the
used as precursors of the natural products were tested against the crystallized structure of a-glucoside hydrolase from Saccharomyces
enzyme, no inhibitory activity was observed, revealing that cerevisiae (aGHY, PDB code 3A4A). The results revealed that
the diketopiperazine subunit had an important impact in the compound 1 bound to the catalytic site of aGHY: the binding energy
31
À1
À1
inhibitory action.
was À6.1 kcal mol while that for acarbose was À8.6 kcal mol
.
The interactions in this site included hydrophobic contacts with
Lys156, Tyr158, Asp242, Val232, Asp233, Phe314, Arg315, Tyr316
3.3 Oral sucrose tolerance test
The inhibitory activity of compounds 1–3 on yeast a-glucosidase and Asn415, and hydrophilic interactions with Ser157, Ser240
prompted us to evaluate their in vivo effect on mammal enzymes and Ser241 (Fig. 3). Hydrogen bonding between compound 1
during a sucrose tolerance test (OSTT) in normal ICR mice. and Ser157 was also observed.
Compound 1 was able to reduce glycaemia levels during the first
3
.5 Oral glucose tolerance test
3
0 minutes of the OSTT; this effect was not dose dependent and
À1
the highest activity was observed at doses 10 and 30 mg kg
Compounds 1 and 3 were tested in an oral glucose tolerance
(Fig. 2, panel A). Compound 3 decreased the postprandial peak test (OGTT), in order to determine their antidiabetic potential
Fig. 2 Effect of cyclodipeptides 1 (panel A) and 3 (panel B) in normoglycaemic ICR mice during an oral sucrose tolerance test (OSTT). VEH: vehicle; ACA:
acarbose. Each point represents the mean Æ SEM of 6–8 mice in each group. *p o 0.01 significantly different from the vehicle group (two-way ANOVA
followed by a Bonferroni post hoc test).
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019

View Article Online
NJC
Paper
Fig. 4 Effect of cyclodipeptides 1 and 3 during an oral glucose tolerance test (OGTT) and intraperitoneal insulin tests. Panels A and B, OGTT in
normoglycaemic mice treated with CDPs 1 and 3, respectively; panel C, OGTT in hyperglycaemic mice treated with CDPs 1 and 3; panel D, insulin
tolerance test in normoglycaemic mice treated with CDPs 1 and 3. VEH: vehicle, MTF: metformin, INS: insulin. Each point or bar represents the mean Æ
SEM of 6–8 mice per group. *p o 0.05 significantly different from vehicle group (ANOVA followed by Bonferroni or Dunnet’s post hoc tests).
independent of the a-glucosidase inhibition. As shown in Fig. 4, not have any effect at the doses tested (panel B). In hyperglycae-
À1
in normoglycaemic mice, cyclodipeptide 1 was able to reduce the mic mice only 1 (10 and 30 mg kg ) had antihyperglycaemic
hyperglycaemia provoked by the oral glucose challenge at a dose action (panel C) suggesting that compound 1 might increase
À1
of 30 mg kg (panel A); on the other hand, cyclopeptide 3 did insulin sensitivity. Indeed, further testing during an insulin
tolerance test revealed that 1 elicited a significant hypoglycaemic
effect. In contrast, peptide 3 reverted the hypoglycaemic action of
insulin (Fig. 4, panel D). The hypoglycaemic effect of compound 1
could be attributed to an improvement in insulin signalling,
modification of insulin secretion and/or an increase in mitochon-
drial bioenergetics, among other mechanisms.
3.6 Effect of compound 1 on insulin signalling
To evaluate whether the hypoglycaemic effect of compound 1
involved an improvement in insulin sensitivity due to enhanced
Fig. 5 Akt phosphorylation in gastrocnemius muscle (A), adipose tissue (B)
and liver (C) of mice treated with compound 1 during an insulin tolerance test
Fig. 6 Effect of compound 1 on insulin secretion in rat insulinoma INS-1E cells
incubated with compound 1 (10 mg mL ) for two hours before the insulin
À1
À1
(0.05 U kg ). Representative blots are shown. The bar graphs represent the
mean Æ SEM of quantitative densitometry of 3 mice.
secretion assay. Each bar is the mean Æ SEM of two independent experiments.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
New J. Chem.

View Article Online
Paper
NJC
Fig. 7 Oxygen consumption rate, basal respiration and ATP production in mouse myoblast cells C2C12 for 1 (18 h incubation). Each bar is the mean Æ
SEM of two or three independent experiments. One-way ANOVA followed by Dunnett’s test (*p o 0.05, **p o 0.01 and ***p o 0.001).
insulin signalling, phosphorylation of Akt was assessed in 4 Conclusions
gastrocnemius muscle, adipose tissue and liver of mice treated
with the vehicle or compound 1 during an insulin tolerance
test. As expected, insulin increased Akt phosphorylation in
gastrocnemius muscle; however, in compound 1-treated mice
These results suggest that compound 1 exerts its antidiabetic
action throughout a mechanism involving a-glucosidase inhibition
and other mechanisms yet to be established. For instance, an
incretin-based mechanism or modulation of other upstream
there was an important reduction of the phosphorylation of the
kinases such as phosphorylation of PTP1B cannot be ruled out
protein (Fig. 5, panel A). In the adipose and liver tissues, the Akt
at the present time. Since compound 1 does not affect insulin
secretion but improves its utilization, it might be a good candidate
phosphorylation level did not change in either treated mouse
group (Fig. 5, panels B and C), probably due to the fact that the
for further investigation for drug development. Moreover, the
mice used were not obese. Our findings were consistent with
presence of DKPs in food products might be useful for preventing
the inhibition of Akt phosphorylation in HeLa cells exerted by a
2
1
diabetes or its progression upon their consumption. The fact that
compound 1 decreased Akt phosphorylation in muscle and mito-
chondrial respiration linked to ATP production highlights also its
potential as an antitumor agent.
mixture of cyclodipeptides obtained from P. aeruginosa PAO1.
Our results ruled out implication of Akt-phosphorylation in the
hypoglycaemic effect of 1.
3.7 Insulin secretion
Next, to find out if the antihyperglycaemic action of compound 1
implicated modulation of insulin secretion, an in vitro cell based
test using rat insulinoma INS-1E was performed. As observed in There are no conflicts to declare.
Fig. 6, 1 was not able to promote insulin secretion in vitro. Our
Conflicts of interest
32
results are in agreement with a previous report in which cyclo(L-His-
L-Pro), a metabolite of the thyrotropin releasing hormone, did not Acknowledgements
33
affect insulin secretion in mouse pancreatic islets.
This work was supported by grants from CONACyT A1-S-11226,
DGAPA IN217516 and IA 204116, and PAIP-UNAM 5000-9140.
A. R. T. thanks CONACyT-Infraestructura 280275. M. L. G.
acknowledges a postdoctoral fellowship from DGAPA-UNAM.
3
.8 Effect of compound 1 on mitochondrial bioenergetics
Hyperglycaemia has also been linked to alterations of mito-
chondrial bioenergetics,
3
4,35
therefore the effect of 1 on mouse
´
The authors also recognize the valuable support of C. Alvarez-
myoblast cell C2C12 mitochondrial energetics was evaluated
over 18 hours. The results indicated that the basal oxygen
consumption rate (OCR) in mouse myoblast cells C2C12 treated
with 1 was significantly reduced. This reduction was primarily
due to a decrease in the OCR associated with ATP production
in a concentration dependent fashion, thus reducing the mito-
chondrial oxidative capacity (Fig. 7). Previous reports showed
that cyclo(L-His-L-Pro) decreased the mitochondrial capacity
due to a reduction in cytochrome C reductase and malic
Toledano. We thank I. Rivero-Cruz, A. V ´a zquez-P ´e rez and
L. Macias-Rosales for their technical assistance.
Notes and references
1 Diagnosis and Classification of Diabetes Mellitus,
American Diabetes Association, Diabetes Care, 2009, 32,
S62–S67.
2 International Diabetes Federation, IDF Diabetes Atlas, Inter-
national Diabetes Federation, 8th edn, Brussels, Belgium,
2017, http://www.diabetesatlas.org.
3
6
enzymes. The results obtained for muscle tissue and mouse
myoblast cells C2C12 suggest that compound 1 might have
important implications in cell proliferation.
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019

View Article Online
NJC
Paper
3
4
5
6
7
B. S. Scales, R. P. Dickson, J. J. LiPuma and G. B. Huffnagle,
Clin. Microbiol. Rev., 2014, 27, 927–948.
N. Cabezas, M. Caramelli, P. Carloni, J. C. Men ´e ndez and
G. Legname, ChemMedChem, 2010, 5, 1324–1334.
M. Mansson, L. Gram and T. O. Larsen, Mar. Drugs, 2011, 9, 21 L. H. Padilla, D. V ´a zquez-Rivera, L. A. S ´a nchez-Briones,
1440–1468.
A. L. D ´ı az-P ´e rez, J. Moreno-Rodr ´ı guez, M. A. Moreno-Eutimio,
V. Meza-Carmen, H. Reyes-De la Cruz and J. Campos-Garc ´ı a,
Molecules, 2017, 22, 1024.
P. G. Brisbane, L. J. Janik, M. E. Tate and R. F. Warren,
Antimicrob. Agents Chemother., 1987, 31, 1967–1971.
L. S. Thomashow, D. M. Weller, R. F. Bonsall and L. S. Pierson, 22 T. Suzuki, J. Yoshida and S. Ono, a-glucosidase Inhibitor,
Appl. Environ. Microbiol., 1990, 56, 908–912.
A. Sajeli-Begum, S. A. Basha, G. Raghavendra, M. V. Kumar,
Japan, PCT/JP2016/054688, WO 2016/133155 Al, August 25th
2016, WIPO I PCT.
Y. Singh, J. V. Patil, Y. Tanemura and Y. Fujimoto, Chem. 23 B. Konrad, A. Dabrowski, M. Szoltysik, P. Marta, Z. Aleksandra
Biodiversity, 2014, 11, 92–100. and C. Jozefa, Int. J. Pept. Res. Ther., 2014, 20, 483–501.
M. Kochar, A. Upadhyay and S. Srivastava, Res. Microbiol., 24 M. Robles-Huizar, S. Torre-Zavala, M. A. Garza-Ramos and
011, 162, 426–435. L. J. Galan-Wong, J. Bacteriol. Res., 2017, 9, 30–36.
W. M. Wuest, E. S. Sattely and C. T. Walsh, J. Am. Chem. Soc., 25 M. Rangel-Grimaldo, I. Rivero-Cruz, A. Madariaga-Maz o´ n,
009, 131, 5056–5057. M. Figueroa and R. Mata, J. Nat. Prod., 2017, 80, 582–587.
0 D. V ´a zquez-Rivera, O. Gonz ´a lez, J. Guzm ´a n-Rodr ´ı guez, A. L. 26 B. Ovalle-Magallanes, O. N. Medina-Campos, J. Pedraza-
8
9
2
2
1
D ´ı az-P ´e rez, A. Ochoa-Zarzosa, J. L ´o pez-Bucio, V. Meza-Carmen
and J. Campos-Garc ´ı a, BioMed Res. Int., 2015, 197608.
1 Q. Guo, D. Guo, B. Zhao, J. Xu and R. Li, J. Nematol., 2007,
Chaverri and R. Mata, Phytochemistry, 2015, 110, 111–119.
27 J. Van den Bossche, J. Baardman and M. P. Winther,
J. Visualized Exp., 2015, 105, 53424.
1
1
1
1
1
1
3
9, 243–247.
28 H. Thajedee, K. Park, S. S. Moon and I. S. Hong, Tetrahedron
Lett., 2010, 51, 1303–1305.
29 P. J. Milne, D. W. Oliver and H. M. Roos, J. Crystallogr.
Spectrosc. Res., 1992, 22, 643–649.
30 Y. P. H. Lee, J. H. Choi, A. Kashima, G. Nakamura and
T. Suzuki, Bull. Korean Chem. Soc., 2014, 35, 2299–2303.
31 K. Yamamoto, H. Miyake, M. Kusunoki and S. Osaki, FEBS J.,
2010, 277, 4205–4214.
32 T. Spacek, J. Santorov ´a , K. Zacharovav ´a , Z. Berkov ´a , L. Hlavat ´a ,
F. Saudek and P. Jezek, Int. J. Biochem. Cell Biol., 2008, 40,
1522–1535.
2 S. T. Beckett, Industrial Chocolate Manufacture and Use,
Blackwell Publishing, USA, 2009.
3 M. Ginz and U. H. Engelhardt, J. Agric. Food Chem., 2000, 48,
3
528–3532.
4 S. Sakamura, K. Furukawa and T. Kasai, Agric. Biol. Chem.,
978, 42, 607–612.
5 A. K. Mishra, J. Choi, S. J. Choi and K. H. Baek, Molecules,
017, 22, E1796.
6 T. R. Welch and R. M. Williams, Nat. Prod. Rep., 2014, 21,
376–1404.
1
2
1
1
1
7 C. Prasad, Peptides, 1995, 16, 151–164.
8 O. S. Kwon, S. H. Park, B. S. Yun, Y. R. Pyun and C. J. Kim,
J. Antibiot., 2001, 54, 179–181.
33 P. Awouters, H. P. Meissner and J. C. Henquin, Diabetes Res.,
1985, 2, 105–110.
34 K. H. Fisher-Wellman and P. D. Neufer, Trends Endocrinol.
Metab., 2012, 23, 142–153.
1
2
9 C. K. Lin, Y. T. Wang, E. M. Hung, Y. L. Yang, J. C. Lee,
J. H. Sheu and C. C. Liaw, Planta Med., 2017, 83, 158–163.
0 M. L. Bolognesi, H. N. A. Tran, M. Staderini, A. Monaco, 36 C. Miyamori, T. Kato, E. Imura, A. Murata, T. Sato, N. Sakura
A. L o´ pez-Cobe n˜ as, S. Bongarzone, X. Biarn ´e s, P. L o´ pez-Alvarado,
and T. Hashimoto, Acta Endocrinol., 1988, 119, 575–581.
35 M. E. Patti and S. Corvera, Endocr. Rev., 2010, 31, 364–395.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
New J. Chem.