Molecular Human Reproduction, Vol.18, No.5 pp. 253– 264, 2012
Advanced Access publication on December 16, 2011 doi:10.1093/molehr/gar080
ORIGINAL RESEARCH
Folic acid and safflower oil
supplementation interacts
and protects embryos from maternal
diabetes-induced damage
R. Higa 1, M. Kurtz 1, M.B. Mazzucco 1, D. Musikant 2, V. White1,
and A. Jawerbaum 1,*
1
Laboratory of Reproduction and Metabolism, CEFYBO-CONICET, School of Medicine, University of Buenos Aires, Paraguay 2155,
1121ABG Buenos Aires, Argentina 2Department of Microbiology, School of Medicine, University of Buenos Aires, Paraguay 2155,
1121ABG Buenos Aires, Argentina
*Correspondence address. E-mail: [email protected]
Submitted on October 21, 2011; resubmitted on November 29, 2011; accepted on December 8, 2011
abstract: Maternal diabetes increases the risk of embryo malformations. Folic acid and safflower oil supplementations have been
shown to reduce embryo malformations in experimental models of diabetes. In this study we here tested whether folic acid and safflower
oil supplementations interact to prevent embryo malformations in diabetic rats, and analyzed whether they act through the regulation of
matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), and nitric oxide (NO) and reactive oxygen species production. Diabetes was induced by streptozotocin administration prior to mating. From Day 0.5 of pregnancy, rats did or did not receive folic acid (15 mg/
kg) and/or a 6% safflower oil-supplemented diet. Embryos and decidua were explanted on Day 10.5 of gestation for further analysis of
embryo resorptions and malformations, MMP-2 and MMP-9 activities, TIMP-1 and TIMP-2 levels, NO production and lipid peroxidation.
Maternal diabetes induced resorptions and malformations that were prevented by folic acid and safflower oil supplementation. MMP-2
and MMP-9 activities were increased in embryos and decidua from diabetic rats and decreased with safflower oil and folic acid supplementations. In diabetic animals, the embryonic and decidual TIMPs were increased mainly with safflower oil supplementation in decidua and with
folic acid in embryos. NO overproduction was decreased in decidua from diabetic rats treated with folic acid alone and in combination with
safflower oil. These treatments also prevented increases in embryonic and decidual lipid peroxidation. In conclusion, folic acid and safflower
oil supplementations interact and protect the embryos from diabetes-induced damage through several pathways related to a decrease in proinflammatory mediators.
Key words: diabetes / pregnancy / folic acid / safflower oil / matrix metalloproteinases
Introduction
Diabetes during pregnancy is associated with increased risks of congenital malformations, mostly cardiac and neural tube defects
(Martinez-Frias, 1994). Their induction occurs during early organogenesis and has been clearly related to the degree of maternal metabolic
imbalance (Kitzmiller et al., 2010). Although still not completely understood, mechanisms of induction of embryo malformations in maternal
diabetes are related to the intrauterine pro-inflammatory environment, reactive oxygen and nitrogen species formation, apoptotic
events and deficiency in arachidonic acid derivatives (Eriksson, 2009;
Jawerbaum and White, 2010; Zabihi and Loeken, 2010). Nitric
oxide (NO) overproduction and the resulting nitrative stress have
been found in both embryos and decidua from diabetic rats during
early organogenesis (Jawerbaum and Gonzalez, 2005; Higa et al.,
2010). The decidua possesses nutritional functions during embryo organogenesis, as well as immunological functions in the regulation of
trophoblast invasion and the formation of the placenta (Parr et al.,
1990; Cohen et al., 2010). Experimental studies have shown that supplementation with safflower oil, which provides linoleic acid, a substrate for the synthesis of arachidonic acid and an endogenous
activator of peroxisome proliferator activated receptors (PPARs), is
capable of reducing maternal diabetes-induced developmental
defects and of regulating pro-inflammatory pathways in the embryo,
the decidua, the fetus and the placenta (Reece et al., 1996; Higa
et al., 2010; Jawerbaum and Capobianco, 2011).
On the other hand, it is well known that folic acid supplementation
highly reduces the induction of congenital malformations in both
& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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254
human pregnancies and experimental diabetic models (Wentzel,
2009). Indeed, in human studies, folic acid supplementation reduces
the induction of congenital defects with doses ranging 0.4 –4 mg/
day by 50 –70% (Goh and Koren, 2008). During development, antioxidant and anti-apoptotic effects of folic acid have been described and
possibly related to their protective effects during embryo organogenesis (van der Put et al., 2001; Wentzel, 2009). As mechanisms involved
in the reduction of congenital malformations of both folic acid and safflower oil supplementations in maternal diabetes are possibly related
to anti-inflammatory effects, we hypothesized that they may cause
beneficial effects by similar mechanisms of action, and thus, that
joint effects of these treatments could be observed. Folic acid supplementation reduces apoptosis and the concentration of isoprostanes,
sensitive markers of oxidative stress, in embryos and yolk sacks
from diabetic rats (Wentzel and Eriksson, 2005; Gareskog et al.,
2006; Zabihi et al., 2007; Jia et al., 2008). On the other hand, safflower
oil supplementation reduces NO production and oxidative stress in
embryos and decidua from diabetic rats (Reece and Wu, 1997; Higa
et al., 2010). Moreover, folic acid has been shown to prevent neural
tube defects caused by excessive NO (Weil et al., 2004). Both NO
and reactive oxygen species (ROS) are positive regulators of matrix
metalloproteinases (MMPs) activity (Ra and Parks, 2007).
MMPs are zinc-dependent endopeptidases capable of cleaving most
components of the extracellular matrix (Mott and Werb, 2004).
MMPs activities have been found to be clearly related to relevant developmental processes such as implantation, decidualization, embryo
organogenesis and placental formation (Vu and Werb, 2000; Cohen
et al., 2010). MMPs are regulated at multiple levels, including transcription, activation of the zymogen and inhibition of their active forms by
the endogenous inhibitors of matrix metalloproteinases (TIMPs)
(Nagase et al., 2006). Both MMP-2 and MMP-9 are MMPs largely
involved in reproductive processes, although they are also markers of
a pro-inflammatory state when produced in excess (Vu and Werb,
2000; Mandal et al., 2003; Huang et al., 2009). We have recently
found an increased activity of MMP-2 and MMP-9 as well as an impaired
MMPs/TIMPs balance that occurred despite increased TIMP-1 and
TIMP-2 concentrations in both embryos and decidua from diabetic
rats during early organogenesis (Higa et al., 2011). MMPs overactivities
are possibly related to the induction of diabetic embryopathy, indeed,
increased MMP-2 and MMP-9 have been involved in the pathogenesis
of several diseases, and are considered markers of a pro-inflammatory
environment (Mandal et al., 2003; Huang et al., 2009). Whether folic
acid and safflower oil treatments alone or in combination can regulate
embryonic MMPs overactivity in maternal diabetes is unknown.
Therefore, this work aimed to address whether maternal treatments
with folic acid and/or safflower oil regulate the activity of MMP-2 and
MMP-9 and modulate TIMP-1 and TIMP-2 in embryos and decidua from
diabetic rats during early organogenesis, as well as to analyze whether
these joint treatments provide greater protection from MMPs, NO and
ROS overproduction and from the induction of congenital malformations.
Materials and Methods
Animals
Albino Wistar rats were bred in the laboratory with free access to commercial rat chow (Asociación Cooperativa Argentina, Buenos Aires,
Higa et al.
Argentina) and water, in a lighting cycle of 14 h light: 10 h dark. Female
rats weighing 200– 230 g were made diabetic with a single i.p. injection
of streptozotocin (55 mg/kg) (Sigma-Aldrich, St Louis, MO, USA) in
citrate buffer (0.05 M, pH 4.5), as previously described (Higa et al.,
2007). Control rats were injected with citrate buffer alone. One week
after diabetes induction, control and diabetic females were mated with
control male rats. Mating was confirmed by the presence of sperm in
vaginal smears and the noon of the day on which the mating was identified
was designated Day 0.5 of pregnancy. The reproductive characteristics of
the diabetic model have been previously described (Jawerbaum and
White, 2010). Glycemia was measured with Accu-Chek reagent strips
and a glucometer (Accu-Chek, Bayer Diagnostics, Buenos Aires,
Argentina) 1 week after streptozotocin administration and on Day 10.5
of gestation. Females showing glycemia values higher than 250 mg/dl
were considered diabetic. The study was carried out in accordance with
the guidelines for the care and use of animals approved by the local institution, according to the ‘Principles of laboratory animal care’ (NIH publication
No. 85–23, revised 1985, http://grants1.nih.gov/grants/olaw/references/
phspol.htm).
Treatments
On Day 0.5 of gestation, both control and diabetic rats were randomized
into four groups (Table I): (A) rats fed ad libitum with the commercial rat
chow that did not receive any treatment except a daily saline solution s.c.
injection (‘non-treated rats’); (B) rats fed ad libitum with the standard diet
supplemented with safflower oil (6% wt/wt) that received a daily saline solution s.c. injection (‘safflower oil-treated rats’); (C) rats fed ad libitum with
the commercial rat chow that received a daily dose of folic acid (15 mg/kg
s.c., vehicle: saline solution) (‘folic acid-treated rats’); and (D) rats fed ad
libitum with the standard diet supplemented with safflower oil (6% wt/wt)
that received a daily dose of folic acid (15 mg/kg s.c., vehicle: saline
solution) (‘safflower oil plus folic acid-treated rats’). All diets met the
nutritional requirements of calories, fat, carbohydrates and proteins for
maintenance and reproduction (Council SoLANNR, 1995), and were
previously used during rat gestation (Higa et al., 2010). The safflower
oil-supplemented diet has similar amounts of carbohydrates, proteins
and calories, and a 6% increase in lipid content, mostly linoleic acid
(226%) as well as palmitic acid (67%) and oleic acid (42%), when
compared with the standard diet (Table I). All treatments were given
until Day 10.5 of gestation, a period corresponding to early embryo
organogenesis in rats (Jawerbaum and Gonzalez, 2005).
Tissue collection and evaluation of embryo
morphology
Animals were euthanized in a CO2 chamber on Day 10.5 of pregnancy,
and the uteri were transferred to Petri dishes with Krebs Ringer Bicarbonate (KRB) solution: 5 mM glucose, 145 mM Na+, 5.9 mM K+, 2.2 mM
22
Ca++ , 1.2 mM Mg++ , 127 mM Cl2, 25 mM HCO2
3 , 1.2 mM SO4 and
32
1.2 mM PO4 . Blood was obtained from the aorta artery and serum
obtained after blood centrifugation and preserved at 2208C for further
determination of glucose, non-esterified fatty acids (NEFA) and triglyceride
concentrations.
By the use of a stereomicroscope and microsurgical dissecting instruments, the balls of decidual tissue were explanted from each uterus, and
gently opened to free the conceptuses. The embryos were dissected
out of the yolk sacs and evaluated morphologically under a stereomicroscope. Decidua were separated from the embryo, the extraembryonic
tissues and the ectoplacental cone. Embryo viability was established by
the presence of a beating heart. The embryos were categorized as morphologically normal or as showing malformations. Malformations were
mostly neural tube defects. Severe malrotation was also observed although
255
Regulation of MMPs in diabetic embryopathy
Table I Experimental design and diet composition of the experimental groups.
Experimental design
Diet composition of the experimental groups
Diet composition
Experimental groups: (1) Non-treated rats; (3) Folic
acid treated rats
Experimental groups: (2) Safflower oil treated rats; (4) Safflower
oil + Folic acid treated rats
Carbohydrates (g/100 g
diet)
50
47
Proteins (g/100 g diet)
25
23
Fat (g/100 g diet)
5
11
Energy content (Kcal/100 g
diet)
324
345
Major fatty acid content (g/100 g diet)
16:0 (palmitic)
0.58
0.97
18:0 (estearic)
0.16
0.25
18:1 (oleic)
1.27
1.81
18:2 (linoleic)
1.99
6.49
18:3 (linolenic)
0.73
0.55
Streptozotocin (STZ), 55 mg/kg or vehicle was given to non-pregnant rats 1 week before mating. Both control and STZ-induced diabetic animals were randomized into four groups on
Day 0.5 of gestation and evaluated on Day 10.5 of gestation.
mainly present concomitantly with embryonic neural tube defects. By using
another group of treated and untreated control and diabetic animals, the
malformation rate was corroborated on Day 12.5 of gestation at which all
non-malformed embryos showed a closed neural tube. Embryos in resorption stages received no further analyses. Viable 10.5 day embryos and their
corresponding decidua were immediately prepared according to the
determinations described below.
Biochemical measurements
Serum triglycerides and NEFA were, respectively, measured by commercial colorimetric kits from Wiener Lab. Rosario, Argentina and Randox,
Antrim, UK.
Analysis of MMPs gelatinase activity
Zymography was performed to evaluate the presence of MMP-2 and
MMP-9 gelatinase activity, as previously described (Pustovrh et al.,
2009). Both MMPs and pro-MMPs were analyzed by zymography, since
the exposure to sodium dodecyl sulfate (SDS) induces changes in
pro-MMPs conformation, which are associated with their activation.
Briefly, this technique evaluates the capacity of MMPs (separated from
other molecular weight proteins through an SDS gel) to degrade the
gelatin which was incorporated in the gel. Three pooled embryos or
one decidua obtained from each rat (n ¼ 7 – 9 rats in each experimental
group) were homogenized in 50 mM Tris, 5 mM CaCl2, 1 mM ZnCl2,
1% Triton X-100. Either 20 mg of protein of embryonic homogenates or
50 mg of protein of decidual homogenates were mixed with loading
buffer (2% SDS), 10% glycerol, 0.1% bromophenol blue, 50 mM Tris –
HCl, pH 6.8) and subjected to a 7.5% SDS – polyacrylamide gel electrophoresis (SDS – PAGE), in which 1 mg/ml gelatin (type A from porcine
skin) had been incorporated. Following electrophoresis, gels were
washed in 2.5% Triton X-100 for 60 min to remove SDS. The gels were
then incubated for 96 h in the case of the embryonic tissue and for 24 h
in the case of the decidual tissue, in 50 mM Tris Buffer pH 7.4, containing
150 mM NaCl and 10 mM CaCl2, at 378C. Then, gels were stained with
Coomassie blue and destained with 10% acetic acid and 30% methanol
in water. The areas of proteolytic activity appeared as negatively stained
bands in the dark background.
The identities of MMPs were based on their molecular weights and a
positive internal control (conditioned medium of human fibrosarcoma
HT-1080 cells). The enzymatic activity was quantified using an image analysis program (ImageJ), and expressed as arbitrary densitometric units,
which were normalized to an internal control. Data are shown as relative
to a value of 1 assigned to the mean values for active MMPs in control
embryos and decidua.
Evaluation of tissue inhibitors of MMPs
To analyze the inhibitory capacity of TIMP proteins in embryos and
decidua from control and diabetic rats, reverse zymography analysis
was performed, as previously described (Pustovrh et al., 2009).
Briefly, this technique evaluates the capacity of TIMPs (separated
256
from different molecular weight proteins through an SDS-gel) to inhibit
the degradation of gelatin by MMPs that were incorporated in the
culture media. Three pooled embryos or one decidua obtained from
each rat (n ¼ 7 – 9 rats in each experimental group) were homogenized
in 50 mM Tris, 5 mM CaCl2, 1 mM ZnCl2, 1% Triton X-100, followed
by a heat extraction (608C) with 50 mM Tris, 0.1 M CaCl2 and
0.15 M NaCl. Either 20 mg of protein from embryonic tissues or
50 mg of protein from decidual tissues were mixed with loading
buffer (2% SDS, 10% glycerol, 0.1% bromophenol blue, 50 mM Tris–
HCl, pH 6.8) and applied to 15% polyacrylamide gels containing 0.1%
SDS and 1 mg/ml gelatin plus 25% conditioned medium of human fibrosarcoma HT-1080 cells, which is a rich source of various MMPs. After
electrophoresis, gels were rinsed twice with 2.5% Triton X-100, and
then incubated at 378C for 24 h in Tris buffer (50 mM Tris – HCl,
0.2 M NaCl, 5 mM CaCl2, pH 8.0). Subsequently, gels were stained
with Coomassie blue and then destained with 10% acetic acid and
30% methanol in water. MMPs provided by the HT-1080 conditioned
medium digested the gelatin within the gel, whereas the TIMPs in the
samples inhibited the MMP action, allowing the identification of the inhibitory capacity of TIMPs as dark bands on a clear background. The
identities of TIMPs were based on their molecular weights, determined
through prestained SDS – PAGE protein standards (prestained Full
Range, GE Healthcare, Buckinghamshire UK), which were run in the
same gel. TIMP activity was quantified using an image analysis
program (ImageJ), and expressed as arbitrary densitometric units.
Data are shown as relative to a value of 1 assigned to the mean
value for TIMPs in control embryos and decidua.
Analysis of NO production
NO production was evaluated in embryos and decidua by the determination of the concentration of its stable metabolites nitrates/nitrites, as previously performed (Higa et al., 2010), by using a commercial assay kit
(Cayman Chemical Co., Ann Arbor, MI, USA). Briefly, four embryos or
one decidua obtained from each rat (n ¼ 7 – 9 rats in each experimental
group) were homogenized in Tris – HCl solution (0.1 mM, pH 7.4) and
an aliquot was separated to determine protein content by the Bradford
method. Nitrates in the supernatant were reduced to nitrites using
nitrite reductase, and total nitrites were then quantified by the Griess reaction. Different amounts of sodium nitrites and nitrates were used as
standards. Optical densities were measured at 540 nm. Results are
expressed as nanomoles/milligram protein.
Evaluation of lipid peroxidation
Lipid peroxidation in embryos was evaluated by measuring 8isoprostanes (8-iso-PGF2a) concentrations, as previously performed
(Higa et al., 2011) by using a commercial enzyme immunoassay kit
(Cayman Chemical Co.). Briefly, three pooled embryos were homogenized in 2 M NaOH (n ¼ 7 – 9 rats in each experimental group) and an
aliquot separated for protein determination by the Bradford method
using a protein assay reagent (Bio-Rad Laboratories Inc.). Samples
were incubated at 458C for 2 h, neutralized by the addition of 2 M
HCl (pH 7.5) and centrifuged at 1000g for 10 min. The assay was developed according to the manufacturer’s instructions. Briefly, the kit uses a
polyclonal antibody against 8-iso-PGF2a, which binds, in a competitive
manner, either the 8-isoprostane in the sample or an 8-iso-PGF2a molecule which has an acetylcholinesterase covalently attached to it. After a
simultaneous incubation, a reagent consisting of acetylthiocholine and
5,5dithio-bis-(2-nitrobenzoic acid) was added. The hydrolysis of acetylthiocholine by acetylcholinesterase yields thiocholine, which reacts
with 5,5dithio-bis-(2-nitrobenzoic acid), generating a yellow color which
Higa et al.
was evaluated on a microplate reader at 412 nm. Results are expressed
as picogram/microgram protein.
Lipid peroxidation in decidua was analyzed by measuring the concentrations of thiobarbituric acid reactive substances (TBARS), as previously performed (Kurtz et al., 2010), a method widely used to assess peroxidation
of fatty acids (Ohkawa et al., 1979). Briefly, 100 mg of tissue was homogenized in 100 mM Tris– HCl buffer, pH 7.6 (n ¼ 7 –9 rats in each experimental group). The homogenate was added with trichloroacetic acid
(40%) and centrifuged at 1.8g for 10 min. The supernatant was added
with an equal volume of thiobarbituric acid (46 mM), and the solution
was heated at 958C for 15 min. Then, the samples were cooled and quantified spectrophotometrically at 530 nm. Malondialdehyde (Sigma-Aldrich)
subjected to the same conditions as the tissue homogenates was used as a
standard. TBARS are expressed as picomoles/milligram protein.
Statistical analysis
Data are presented as the mean + standard error. Groups were compared by two-way analysis of variance (ANOVA) in conjunction with Bonferroni’s test. For malformation and resorption rates, data were compared
using x2 contingency tests. In all cases, differences were considered statistically significant at P , 0.05.
Results
Resorption and malformation rate in diabetic
rats treated with safflower oil and/or folic
acid during pregnancy
All diabetic animals, non-treated or treated with safflower oil (6%)
and/or folic acid (15 mg/kg) during pregnancy showed increased
serum glucose, NEFA and triglycerides on Day 10.5 of gestation
when compared with their respective controls (Table II). No
changes in serum glucose and NEFA were found when treated and
non-treated diabetic rats were compared. A decrease in serum triglycerides concentration was found in both the control P , 0.01) and
diabetic (P , 0.05) groups treated with folic acid alone but not in
combination with safflower oil when compared with the non-treated
groups (Table II).
When we analyzed the resorption rate, which was increased 5.7
times in the non-treated diabetic animals when compared with controls (P , 0.001), we found that the three diabetic animal treated
groups showed a decreased resorption rate when compared with
the non-treated diabetic animals (safflower oil 64.4%, P , 0.001,
folic acid 84.2%, P , 0.001, safflower oil plus folic acid 80%,
P , 0.001, Table II).
When we evaluated the malformation rate, which was increased 13
times in the non-treated diabetic animals when compared with controls (P , 0.001), we found that both safflower and folic acid treatments were able to significantly decrease the malformation rate in
diabetic animals (57.5%, P , 0.05 and 62.4%, P , 0.05, respectively).
Of note, only the combination of both safflower and folic acid treatments was able to decrease the malformation rate in diabetic rats
to values similar to those found in the control animals (84%,
P , 0.01, Table II).
257
Regulation of MMPs in diabetic embryopathy
Table II Maternal serum glucose, NEFA and triglycerides concentrations, as well as resorption and malformation rates in
control and diabetic rats treated or not with safflower oil and/or folic acid.
No treatment
Safflower oil
................................... ..................................
Control
Diabetic
Control
Diabetic
Safflower oil 1 folic acid
Folic acid
................................... ...................................
Control
Diabetic
Control
Diabetic
.............................................................................................................................................................................................
Maternal glycemia (mg/dl)
Maternal Serum NEFA (mM)
Maternal triglyceridemia (g/l)
105 + 15a
439 + 27b
a
0.64 + 0.05
a
1.09 + 0.12
ac
90 + 40a
bc
0.80 + 0.05
b
1.69 + 0.21
b
356 + 25b
a
0.64 + 0.05
a
0.86 + 0.09
ac
99 + 28a
b
0.85 + 0.05
b
1.95 + 0.38
c
379 + 18b
a
0.67 + 0.03
c
0.54 + 0.06
a
97 + 36a
bc
0.83 + 0.07
a
1.06 + 0.10
ac
438 + 33b
ac
0.92 + 0.09b
ac
1.79 + 0.20b
0.69 + 0.05
0.80 + 0.18
a
Resorption rate: Embryo
resorptions/total
implantation sites (%)
10/173
(5.8%)
51/154
(33.1%)
13/142
(9.1%)
19/161
(11.8%)
5/126
(3.9%)
7/135
(5.2%)
4/126
(3.17%)
8/121ac
(6.6%)
Malformation rate:
Malformed embryos/ total
viable embryos (%)
2/163a
(1.2%)
17/103b
(16.5%)
1/129a
(0.8%)
10/142c
(7.0%)
1/121a
(0.8%)
8/128c
(6.2%)
1/122a
(0.8%)
3/113ac
(2.6%)
Serological data are shown as means + SEM and evaluated by two-way ANOVA in conjunction with Bonferroni’s test. Resorption and Malformation rates are shown as percentage and
evaluated by x2 contingency tests. Values were obtained from 10 to 13 rats in each experimental group. Different letters denote significant differences (P , 0.05) between groups.
Embryonic and decidual MMPs activities in
diabetic rats treated with safflower oil and/or
folic acid during pregnancy
Considering the relevance of the embryonic and decidual MMP-2 and
MMP-9 in embryo and decidual remodeling throughout development,
and the overactivity of these MMPs previously found in diabetic
animals (Vu and Werb, 2000; Cohen et al., 2010; Higa et al., 2011),
we analyzed MMP-2 and MMP-9 activities in embryos and decidua
from diabetic rats treated with safflower oil (6%) and/or folic acid
(15 mg/kg) during pregnancy.
We found that MMP-2, which was overactivated in the embryos
from diabetic rats in its active (P , 0.01) and proenzyme forms
(P , 0.001), was decreased in the embryos from safflower oil
(MMP-2: P , 0.01, proMMP-2: P , 0.001) and safflower oil plus
folic acid (MMP-2: P , 0.05, proMMP-2: P , 0.001) -treated diabetic
rats when compared with the non-treated diabetic group (Fig. 1). No
changes in embryonic MMP-2 activity were found when diabetic
animals treated with folic acid alone were compared with the nontreated ones. Embryos from control animals treated with safflower
oil also showed a decrease in the active form of MMP-2 when compared with the non-treated control group (P , 0.001), and no other
changes in embryonic MMP-2 were observed in the control groups
(Fig. 1).
On the other hand, as regards MMP-9, in the embryos we detected
only its active form, which was increased in the embryo from nontreated diabetic rats (P , 0.001), and decreased in embryos from diabetic rats treated with safflower oil alone (P , 0.05) and in combination with folic acid (P , 0.01), but not with folic acid
alone, compared with the non-treated diabetic group (Fig. 2).
When we analyzed the gelatinolytic activity of MMP-2 in the
decidua, we did not detect changes in its active form in any of
the control and diabetic experimental groups evaluated. Differently,
proMMP-2, which was increased in the decidua from non-treated
diabetic animals (P , 0.001), was decreased in the decidua from
safflower oil plus folic acid diabetic-treated animals (P , 0.001),
but not when either safflower oil or folic acid were given
alone (Fig. 3).
On the other hand, as regards MMP-9 gelatinolytic activity, we
found that its active form was enhanced in the decidua from nontreated diabetic animals (P , 0.001), and also in this case, only the
combined treatment with safflower oil and folic acid was able to decrease MMP-9 activity in diabetic rats when compared with the nontreated diabetic group (P , 0.01; Fig. 4).
Embryonic and decidual TIMPs in diabetic
rats treated with safflower oil and/or folic
acid during pregnancy
TIMP-1 and TIMP-2 are endogenous MMPs inhibitors involved in
embryo development and decidual remodeling, previously found
increased in embryos and decidua from diabetic rats (Alexander
et al., 1996; Higa et al., 2011). Therefore, we analyzed TIMP-1 and
TIMP-2 MMPs inhibitory capacity in embryos and decidua from diabetic rats treated with safflower oil (6%) and/or folic acid (15 mg/kg)
during pregnancy.
We found that maternal treatments with safflower oil (P , 0.05),
folic acid (P , 0.001) and their combination (P , 0.05) led to
further increases in TIMP-1 inhibitory capacity in embryos from diabetic animals compared with the non-treated diabetic group
(Fig. 5B). In embryos from control rats, only the folic acid treatment
alone led to increases in TIMP-1 inhibitory capacity compared with
the non-treated group (P , 0.001).
On the other hand, TIMP-2 inhibitory capacity was found further
increased in the embryos from diabetic rats treated with safflower
oil and folic acid alone (P , 0.05 and P , 0.01, respectively), although
not in combination, when compared with the non-treated diabetic
group (Fig. 5C).
When TIMPs were analyzed in the decidua, we found that both in
the control and in the diabetic group, the only treatment that induced
significant changes was the safflower oil treatment, which led to
increases in decidual TIMP-1 and TIMP-2 inhibitory capacity in the
control (TIMP-1: P , 0.001, TIMP-2: P , 0.05) and the diabetic
(P , 0.01) groups, respectively compared with the non-treated
control and diabetic groups (Fig. 6).
258
Higa et al.
Figure 1 MMP-2 activities evaluated in embryos from treated and non-treated control and diabetic rats on Day 10.5 of gestation. (A) Representative zymogram exhibiting embryonic MMP-2 activities. (B) Densitometric analysis of proMMP-2 activity. (C) Densitometric analysis of MMP-2 activity. Values are means + SEM. n ¼ 7 – 9 rats in each experimental group. Two-way ANOVA in conjunction with Bonferroni’s test was performed.
Different letters denote significant differences (P , 0.05) between groups.
Embryonic and decidual NO production and
lipid peroxidation in diabetic rats treated
with 6% safflower oil and/or folic acid during
pregnancy
As NO and ROS are closely related to diabetes-induced embryopathy
and are capable of activating MMPs (Jawerbaum and Gonzalez, 2005;
Ra and Parks, 2007), nitrates/nitrites (an index of NO production)
and lipid peroxidation (an index of ROS-induced damage), were evaluated in embryos and decidua from control and diabetic rats treated
with safflower (6%) and folic acid (15 mg/kg) alone or in combination
during pregnancy. We found that embryonic NO production, which
was enhanced in the non-treated diabetic rats when compared with
controls (P , 0.001), was significantly decreased by the safflower oil
treatment (P , 0.05), although not in combination with folic acid
(Fig. 7A).
The concentration of 8-isoprostane, which was increased in the
embryos from non-treated diabetic rats when compared with nontreated controls (P , 0.05), was significantly decreased in embryos
from diabetic rats treated with folic acid either alone (P , 0.001) or
in combination with safflower oil (P , 0.001), although not in the diabetic group treated only with safflower oil, when compared with the
non-treated diabetic group (Fig. 7B). In control rats, safflower oil treatment led to an increase in embryonic 8-isoprostane concentrations
and folic acid treatment led to a decrease in embryonic 8-isoprostane
concentrations when compared with the non-treated group, although
no other changes were detected in embryonic NO production and
lipoperoxidation.
Figure 2 MMP-9 activities evaluated in embryos from treated and
non-treated control and diabetic rats on Day 10.5 of gestation. (A)
Representative zymogram exhibiting embryonic MMP-9 activities.
(B) Densitometric analysis of MMP-9 activity. Values are means +
SEM. n ¼ 7 – 9 rats in each experimental group. Two-way ANOVA
in conjunction with Bonferroni’s test was performed. Different
letters denote significant differences (P , 0.05) between groups.
On the other hand, in the decidua, NO production, which was
enhanced in the non-treated diabetic group (P , 0.001), was highly
decreased by the folic acid treatment alone (P , 0.001) and in
259
Regulation of MMPs in diabetic embryopathy
Figure 3 MMP-2 activities evaluated in decidua from treated and non-treated control and diabetic rats on Day 10.5 of gestation. (A). Representative
zymogram exhibiting decidual MMP-2 activities. (B) Densitometric analysis of proMMP-2 activities. (C). Densitometric analysis of MMP-2 activity.
Values are means + SEM. n ¼ 7 – 9 rats in each experimental group. Two-way ANOVA in conjunction with Bonferroni’s test was performed. Different
letters denote significant differences (P , 0.05) between groups.
(P , 0.001), folic acid (P , 0.001) and their combination (P ,
0.001) when compared with the non-treated diabetic group
(Fig. 8B). Decreases in lipid peroxidation were also evident in the
decidua from control rats treated with safflower oil in combination
with folic acid when compared with the non-treated control group
(P , 0.05, Fig. 8B).
Discussion
Figure 4 MMP-9 activities evaluated in decidua from treated and
non-treated control and diabetic rats on Day 10.5 of gestation. (A)
Representative zymogram exhibiting decidual MMP-9 activities. (B)
Densitometric analysis of MMP-9 activities. Values are means + SEM.
n ¼ 7 – 9 rats in each experimental group. Two-way ANOVA in conjunction with Bonferroni’s test was performed. Different letters
denote significant differences (P , 0.05) between groups.
combination with the safflower oil treatment when compared with the
non-treated diabetic group (P , 0.001; Fig. 8A). Decidual lipid peroxidation, evaluated through the measurement of TBARS, which was
increased in the decidua from non-treated diabetic rats (P , 0.05),
was decreased in the diabetic animals treated with safflower oil
In this study, we demonstrated that joint treatments with folic acid and
safflower oil of diabetic pregnant rats are more effective in preventing
neural tube defects than each treatment alone. As the most important
findings, we observed that these maternal treatments induced beneficial changes on embryonic and decidual MMPs and TIMPs activities,
which together with the regulation of NO and ROS production, are
likely to be involved in the prevention of diabetic embryopathy.
Previous studies addressing folic acid or safflower oil supplementation in diabetic embryopathy have shown the capacity of these treatments to reduce embryo resorptions and malformations (Wentzel
et al., 2005; Reece et al., 2006; Oyama et al., 2009; Higa et al.,
2010). Indeed, it is well known that folate supplementation for the
general population can reduce the incidence of congenital malformations by 50 –70% (Goh and Koren, 2008). In this work, we found
that diabetic rats treated with folic acid or safflower oil during pregnancy showed, respectively, a 57 –62% reduction in the induction of
neural tube defects, while the combined treatment led to an 84% reduction of embryo malformations. This suggests a possible useful combination for further improving the prevention of congenital
malformations in maternal diabetes. A limitation of this study is that
we did not test lower doses of folic acid. Indeed, the folic acid
260
Higa et al.
Figure 5 TIMP-1 and TIMP-2 inhibitory capacity in embryos from treated and non-treated control and diabetic rats on Day 10.5 of gestation. (A)
Representative reverse zymogram exhibiting embryonic TIMP-1 and TIMP-2 inhibitory capacity. (B) Densitometric analysis of TIMP-1 inhibitory capacity. (C) Densitometric analysis of TIMP-2 inhibitory capacity. Values are means + SEM. n ¼ 7 – 9 rats in each experimental group. Two-way ANOVA
in conjunction with Bonferroni’s test was performed. Different letters denote significant differences (P , 0.05) between groups.
Figure 6 TIMP-1 and TIMP-2 inhibitory capacity in decidua from treated and non-treated control and diabetic rats on Day 10.5 of gestation. (A)
Representative reverse zymogram exhibiting decidual TIMP-1 and TIMP-2 inhibitory capacity. (B) Densitometric analysis of TIMP-1 inhibitory capacity.
(C) Densitometric analysis of TIMP-2 inhibitory capacity. Values are means + SEM. n ¼ 7 – 9 rats in each experimental group. Two-way ANOVA in
conjunction with Bonferroni’s test was performed. Different letters denote significant differences (P , 0.05) between groups.
Regulation of MMPs in diabetic embryopathy
261
Figure 7 NO production and 8-isoprostane concentrations in embryos from treated and non-treated control and diabetic rats on Day 10.5 of
gestation. (A) Embryonic nitrates/nitrites, an index of NO production. (B) Concentration of 8-isprostanes, a marker of oxidative stress. Values
are means + SEM. n ¼ 7 – 9 rats in each experimental group. Two-way ANOVA in conjunction with Bonferroni’s test was performed. Different
letters denote significant differences (P , 0.05) between groups.
Figure 8 NO production and lipid peroxidation in decidua from treated and non-treated control and diabetic rats on day 10.5 of gestation. (A)
Decidual nitrates/nitrites, an index of NO production. (B) Concentration of TBARS, an index of lipid peroxidation. Values are means + SEM.
n ¼ 7 – 9 rats in each experimental group. Two-way ANOVA in conjunction with Bonferroni’s test was performed. Different letters denote significant
differences (P , 0.05) between groups.
doses that should be used in diabetic gestations are still unclear
(Mathiesen et al., 2011) and recent results have shown possible
adverse effects of high doses of folates in embryo development
(Pickell et al., 2011).
The diabetic model used has been previously characterized during
gestation in our laboratory (Higa et al., 2010; Jawerbaum and
White, 2010), and has an increased energy intake (Control:
65+2 kcal/day, Diabetic: 89+5 kcal/day, P , 0.001) and a
decreased weight gain during Days 0.5–10.5 of pregnancy (Control:
30+2 g, Diabetic: 26+1 g, P , 0.001) when compared with controls. As metabolic changes, we found that serum glucose, triglycerides
and NEFA were increased in diabetic animals when compared with
controls, and that folic acid alone (although not in combination with
safflower oil) led to a decrease in triglyceridemia. Studies performed
in pregnant and non-pregnant animals have reported that supplementation with folic acid reduces triglyceridemia and that deficiency in
methyl donors increases tissue triglyceride concentrations (McNeil
et al., 2008; Ojeda et al., 2008).
As the main novel findings of this work, we found that both safflower and folic acid treatments have the capacity to regulate MMPs
and TIMPs activities. These activities were evaluated separately, and
down-regulation of MMPs and/or up-regulation of TIMPs were
observed in embryos and decidua from diabetic rats treated with safflower and/or folic acid. We found differences between the capacity
of the two treatments alone and in combination to regulate MMPs
and TIMPs in embryonic and decidual tissues from diabetic rats,
showing a very different pattern of expression and regulation of
MMPs and TIMPs in the embryo and its surrounding decidua.
262
Indeed, the MMPs evaluated were highly decreased by the maternal
treatment with safflower oil alone and in combination with folic acid
in the embryos, although only with the combined treatment in the
decidua. This may suggest that maternal treatments capable of ameliorating the pro-inflammatory environment in both the embryo and the
decidua during early organogenesis can lead to greater beneficial
effects in the embryo from diabetic rats.
Diets supplemented with safflower oil are enriched in linoleic acid,
an essential n-6 polyunsaturated fatty acid (PUFA) that is transferred
through the yolk sac and the placenta for proper embryonic and
fetal development, capable of activating the three PPAR isotypes
(Xu et al., 2007). Although n-3 PUFAs, present in high concentrations
in fish oil, can also activate PPARs and possess anti-inflammatory
effects, they could lead to a decrease in embryonic arachidonic acid
concentrations, highly required during embryo organogenesis
(Amusquivar et al., 2000; Jawerbaum and Gonzalez, 2005).
There are relevant differences in PPARs expression in embryonic
and decidual tissues, as during early organogenesis the embryo
express only the PPARd isotype while maternal tissues expresses
the three PPAR isoforms (Higa et al., 2007; Abbott, 2009), a difference that may be involved in the different responses observed in decidual and embryonic tissues and that would require further
evaluation. Although several works have found that activation of
PPARg is involved in the restoration of MMPs/TIMPs balance in
various tissues (Hua et al., 2009; Pustovrh et al., 2009), there are
still few data reporting MMPs/TIMPs regulatory pathways by the activation of PPARd, a relevant PPAR isotype during embryo organogenesis (Higa et al., 2007; Kim et al., 2009). Folic acid treatments have also
shown to be capable of regulating MMPs/TIMPs balance in different
tissues (Guo et al., 2006; Qipshidze et al., 2010). Indeed, besides
the complex and pleiotropic functions and mechanisms of action of
folates during development (van der Put et al., 2001), a close interaction between PPARs and folates regulatory pathways has been
described and related to their anti-inflammatory effects (Hayden and
Tyagi, 2004). Therefore, the interaction between folic acid and safflower oil treatments may be the result of both common and independent effects of each of the treatments. In agreement, in this
study we found that the folate treatment alone, although not in combination with safflower oil, induced changes in triglycerides concentrations that could benefit embryo development in maternal diabetes.
NEFA concentrations were unchanged with the performed treatments, although increased in the sera from diabetic rats. On the
other hand, in this work we found that relevant post-transductional
mechanisms capable of regulating MMPs activities were differentially
regulated in embryos and decidua by the safflower and folic acid treatments alone and in combination. Indeed, both the safflower oil and
folic acid treatments were able to increase embryonic TIMPs while
only the safflower oil treatment led to increased TIMPs in the
decidua. The observed increased TIMPs concentrations suggest an
increased capacity to inhibit MMPs overactivity in both the embryo
and the decidua. TIMPs concentrations are up-regulated by PPARg activation in various tissues (Hua et al., 2009; Pustovrh et al., 2009), and
there are some recent reports of the capacity of folic acid to
up-regulate tissue TIMPs concentrations (Qipshidze et al., 2010).
TIMPs are relevant due to their capacity to inhibit MMPs activity,
but also possess several functions related to cell proliferation and differentiation (Stetler-Stevenson, 2008). Therefore, the impact of the
Higa et al.
changes observed in embryonic and decidual TIMPs inhibitory capacity
on the treated diabetic animals will require further research to be fully
elucidated.
Various previous works have addressed the involvement of NO in
neural tube formation and the relevance of NO overproduction in diabetic embryopathy (Plachta et al., 2003; Jawerbaum and Gonzalez,
2005). Besides, the protective effects of folic acid have been found
related to the prevention of NO-derived oxidative molecules in
various tissues (Weil et al., 2004; McCarty et al., 2009). Nevertheless,
in this study we found that in maternal diabetes, folic acid supplementation was not able to reduce NO production in the embryos, although
it clearly regulated NO production in the decidua. In contrast, safflower
oil decreased NO production in embryos and not in decidua, suggesting specific embryonic and decidual effects of safflower oil and folate
treatments on NO production both possibly helping to prevent
diabetes-induced developmental defects. On the other hand, supplementation with folic acid alone and in combination with safflower oil
was able to reduce ROS formation in the embryos, while all treatments
were able to reduce lipoperoxidation in decidua from diabetic animals.
Previous works demonstrated a clear involvement of oxidative stress in
diabetic embryopathy and showed the capacity of folic acid treatments
to reduce embryonic oxidative stress (Eriksson, 2009; Wentzel, 2009;
Zabihi and Loeken, 2010). Our results suggest that the combined treatment of folic acid and safflower oil could constitute an effective antioxidant treatment in maternal diabetes, with regulatory effects on both
the embryo and the decidua. Nevertheless, it should be taken into
account that fat requirements are lower in rats than in humans
(Council SoLANNR, 1995), and thus difficulties may arise in the translation from animals to human studies.
In conclusion, we found that safflower oil and folic acid treatments
alone and in combination lead to the regulation of MMPs and TIMPs
activities, together with changes in the formation of NO and ROS, suggesting anti-inflammatory and antioxidant effects helpful in the prevention of diabetic embryopathy. Since in most measurements the
combined treatment of safflower oil and folic acid was the most effective and led to a greater reduction in embryo malformations, this combination may be considered for further evaluation in clinical trials.
Acknowledgements
We would like to thank Dr Evangelina Capobianco for support in
serum determinations.
Authors’ roles
R.H. was involved in experimental design and procedures, data analysis and interpretation; M.K., M.B.M., D.M. played a role in experimental procedures and manuscript revision; V.W. took part in Data
analysis and interpretation; A.J. was involved in Concept and design,
interpretation and article draft.
Funding
This work was supported by grants from Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (PIP 2010/0002); and Agencia de
Promoción Cientı́fica y Tecnológica of Argentina (PICT 2010-00034).
Regulation of MMPs in diabetic embryopathy
Conflict of interest
None declared.
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