M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL 6 2 ( 2 0 13 ) 44 2–4 45

Available online at www.sciencedirect.com

Metabolism
www.metabolismjournal.com

Maternal hypertension induced by NO blockade does not

program adult metabolic diseases in growth-restricted
rat fetuses

Laura Butruille a,⁎, Sylvain Mayeur a , Emmanuelle Moitrot a , Laurent Storme a ,

Claude Knauf b , Jean Lesage a , Philippe Deruelle a
a
Université Lille Nord de France, Unité Environnement Périnatal et Croissance, EA 4489, Faculté de Médecine, Pôle Recherche, IFR 114,
59045 Lille
b
Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Team 3, INSERM U1048, 31432 Toulouse, France

A R T I C LE I N FO AB S T R A C T

Article history: Objective. Preeclampsia is a frequent and potentially lethal placental insufficiency
Received 16 March 2012 pathology causing maternal hypertension and proteinuria, as well as a high rate of intra-
Accepted 11 September 2012 uterine growth retardation (IUGR) in offspring. Reduced nitric oxide (NO) production may
play a role in the mechanisms of this disease. As exposure to adverse early life environment
Keywords: and IUGR has been proposed to increase cardiometabolic diseases risk, we investigated in
Maternal hypertension rats the effects of maternal NO blockade on growth and metabolic phenotype of offspring.
IUGR Material and Methods. Osmotic pumps were implanted in pregnant rats at E17 and
Fetal programming diffused saline or L-NAME (50 mg/day), a nitric oxide synthesis inhibitor. At birth, IUGR male
Brown adipose tissue newborns without limb defects were selected. Body growth, feeding behavior and glucose
Catch up growth tolerance were evaluated in offspring. Organs weights, plasma level of several metabolic
hormones and genes expressions were determined in fasted 9 month-old rats.
Results. L-NAME mothers had elevated blood pressure at E20. Male offspring from
L-NAME mothers had a markedly reduced birth weight and developed postnatal catch-up
growth during lactation. Some L-NAME newborns presented some limb defects but were not
selected in this study (1/3 of all pups). Improved glucose tolerance and hyperphagia after
fasting were found in 3-month-old L-NAME rat but not thereafter. In 9-month-old L-NAME
rats, a moderate increase of food intake during the light phase and, after fasting, an
augmentation of plasma insulin and a reduction of brown adipose tissue (BAT) deposit were
found associated with an increased expression of UCP-1 mRNA in this tissue.
Conclusions. Despite IUGR and postnatal catch up growth, male rats exposed to L-NAME
did not develop metabolic diseases when limb defects were not induced by L-NAME. We
postulate that maternal hypertension during late gestation is not a major ‘programming’
metabolic factor for offspring.
© 2013 Elsevier Inc. All rights reserved.

Abbreviations: CTRL, controls; L-NAME, Nω-Nitro L-Arginine methyl ester; IUGR, intrauterine growth retardation; NO, nitric oxide; NOS,
nitric oxide synthase; WAT, white adipose tissue; BAT, brown adipose tissue; NPY, neuropeptide Y; POMC, proopiomelanocortin; UCP1,
uncoupling protein 1; OGTT, oral glucose tolerance test; PND, postnatal day.
⁎ Corresponding author. UPRES EA 4489, IFR 114-IMPRT, Faculté de médecine Pôle Recherche, 1, place de Verdun, 59045 Lille Cedex.
Tel.: +33 0 622355763; fax: +33 0 320 44 63 11.
E-mail address: laura.butruille@hotmail.fr (L. Butruille).

0026-0495/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.metabol.2012.09.006
 M ET ABOL I SM CL IN I CA L A N D EX PE RI ME N TA L 6 2 ( 2 0 13 ) 44 2–4 4 5
1. Introduction
Several epidemiological and clinical studies have revealed an
inverse relationship between a reduced birth weight and an
increased risk to develop a metabolic syndrome in adulthood
associating obesity, type 2 diabetes and cardiovascular dis-
eases [1–3]. Yallampalli and Garfield have shown that con-
tinuous infusion of L-NAME, an inhibitor of NOS, caused
hypertension in pregnant rats, decreased litter size and in-
duced a drastic fetal growth restriction by ~40% and limb
defects in some fetuses [4]. In this model, there is little infor-
mation about the long-term outcome of the offspring. We
hypothesized that L-NAME-induced IUGR may be associated
with development of metabolic disorders in adulthood [5,6]. To
answer this question, we evaluated growth, feeding behavior,
glucose tolerance and body composition in IUGR L-NAME
male rats without limb defects from birth until adulthood.
2. Material and methods
Pregnant Wistar Han rats (Charles River) were maintained
under standard conditions. At day 17 of pregnancy (E17), os-
motic minipumps (Alzet 2ML1, Charles River) were implanted
subcutaneously during isoflurane anesthesia and delivered
continuously L-NAME 50 mg/day or saline (CTRL). Some
L-NAME newborns presented some limb defects at birth but
were not studied thereafter. Systolic, diastolic and mean blood
pressures were measured at E20 by the tail-cuff plethysmog-
raphy method (BP-2000 Series II, Bioseb).
At postnatal day 2(PND 2), pups were adopted by nursing
mothers, weighed, and litter size was adjusted to 8 pups per
dam (n =15 for CTRL and n=14 for L-NAME).
2.1. Food intake
Basal food intake was calculated during 4 consecutive days,
separating diurnal and nocturnal consumption at 3, 6 and
9 months. Food consumption after 24 h of fasting was mea-
sured during the first, second, and third hour after reintroduc-
tion of food in cages.
2.2. OGTT
After an overnight fasting rats received 2 g glucose/kg with
an oral cannula. Blood glucose was read on glucometer (Accu-
Chek Performa, Roche) and blood samples were collected
from the tail vein 5 min before (time 0) and 30, 60, 90 and
120 min later.
2.3. Plasma and tissue collections
At 9 months, fasted rats were killed by decapitation. Blood
samples were collected. Perirenal and epididymal white adi-
pose tissues(WAT), brown adipose tissue(BAT), heart, lungs,
kidneys, adrenal glands and thymus were removed, weighed,
and frozen in liquid nitrogen. Plasma insulin (Mercodia, 10-
1251-01), leptin (Bertin Pharma, A05176) and apelin (Phoenix
Pharmaceutical, EK-057-23) were measured using EIA kits.
443
Lipid hormones were measured using a VetTest chemistry
analyzer (IDEXX Laboratories, Model 8008) with triglycerides
and cholesterol adapted panels.
2.4. mRNAs quantifications
Quantification of the relative expression level of mRNA in
the hypothalamus and intrascapular BAT was performed by
qRT-PCR. RNA was extracted and purified using TRIzol
reagent (Invitrogen, Life Technologies) and an equal amount
of total RNA (1 μg) was used for cDNA synthesis (Thermo-
script™, Invitrogen, Life Technologies, France). The amount of
target relative to a calibrator was computed by 2−ΔΔCT. Primers
sequences are detailed in supplementary data.
2.5. Statistical analysis
All data are presented as means±SEM. Samples were anal-
yzed using Student's t-tests and two-way ANOVA followed
by a Newman–Keuls (NK) post-hoc.
3. Results
There was no significant difference in the weight gain during
the last week of gestation between L-NAME and CTRL
mothers. NOS inhibition by L-NAME diffusion resulted in
elevated systolic, diastolic and mean arterial blood pressure at
day 20 of gestation (127±2 vs 144±3 for systolic blood pressure
for CTRL and L-NAME respectively, P<0.001). At birth (E21),
36% of L-NAME rats presented some limb defects but were not
studied thereafter. Necrosis could be observed in one, two or
all limbs of pups, and sometimes affected the tail. Whatever
the necrosis localization, all pups with limb defects were
excluded. At PND2, body weight of L-NAME male rats was
reduced by ~20% compared to CTRL (6.17±0.26 vs 5.12±0.16;
Fig. 1A, P<0.001) but they caught up the body weight of
controls at PND10. Up to 9 months, no significant differences
were observed between groups.
At 3 months, L-NAME offspring had a decreased response
to glucose bolus at T30 and T60 compared to controls (161±6 vs
139±3 mg/dL, P<0.01 at T30 and 166±5 vs 152±3 mg/dL, P<0.05
at T60, for CTRL and L-NAME respectively; Fig. 1B). Plasma
insulin level was slightly diminished (17.2±3.8 vs 12.7±4.9,
CTRL vs L-NAME), and glucose levels were similar between
groups at the age of 6 and 9 months.
Basal food intake was similar in both groups at 3 months
(Fig. 1C), but after 24 h of fasting an increased food intake
was observed in L-NAME rats (1.7±0.2 vs 2.3±0.1, P<0.01 for
the first hour; 2.6±0.1 vs 3.0±0.2, P<0.05 for the second hour
and 2.8±0.2 vs 3.5±0.2, P<0.01 for the third hour, for CTRL
and L-NAME respectively, Fig. 1D). In contrast, L-NAME
9 month-old rats showed higher diurnal food intake com-
pared to CTRL (1.15±0.06 vs 1.40±0.07, P<0.05, Fig. 1E) but no
difference was found after 24 h of fasting (Fig. 1F).
As shown in Table 1, a reduced (−21%) weight of BAT was
reported for 9 month-old L-NAME rats. Blood glucose, leptin
and apelin plasma levels and numerous organs' weight were
similar between groups. Triglycerides and cholesterol plasma
levels were decreased in L-NAME rats (P<0.05). Although
444 M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL 6 2 ( 2 0 13 ) 44 2–4 45

A B C D

E F G H

Fig. 1 – A. Body weight curves of male rat offspring of CTRL and L-NAME dams before weaning. B. Time course of plasma glucose
after OGTT in 3 month-old CTRL and L-NAME rats. C. Basal feeding behavior during light phase, dark phase and daily at
3 months of age. D. Time course of refeeding after 24 h of starvation during the first 3 h. Weight related food intake was
measured in 3 month-old CTRL and L-NAME rats. E. Basal feeding behavior during light phase, dark phase and daily at 9 months
of age. F. Time course of refeeding after 24 h of starvation during the first 3 h. Weight related food intake was measured in
9 month-old CTRL and L-NAME rats. G. Effects of chronic nitric oxide synthase inhibition by L-NAME on mRNA expression in
9 month-old male rats of hypothalamic NPY and POMC. H. Effects of chronic nitric oxide synthase inhibition by L-NAME on
mRNA expression in 9 month-old male rats UCP1 in brown adipose tissue. Values are means±SEM (n=7–8 per group), * P<0.05
** P<0.01 *** P<0.001.

L-NAME animals displayed a slight hyperinsulinemia (16.3±2.7 In brown adipose tissue, UCP1 expression was increased
vs 25.7±1.8 μU/mL for CTRL and L-NAME respectively, P<0.05), by 1.7 fold in L-NAME group compared to CTRL (Fig. 1H). In
no differences in basal blood glucose levels was noted. the hypothalamus, both NPY and POMC mRNA expression
levels were not modified between L-NAME and CTRL groups
(Fig. 1G).
Table 1 – Effects of maternal treatment with L-NAME on
weight and morphometric and biological parameters, at
9 months after 16 h of fasting. 4. Discussion
Controls L-NAME
In the present study, we showed that L-NAME offspring
Birth weight, g 6.17±0.3 5.12±0.2 ⁎⁎
had a reduced birth weight compared to CTRL and caught
Body weight, g 416±15 433±16
up with the growth of CTRL at PND10. At 3 months of age,
Perirenal WAT, mg/100 g of body weight 2967±342 2882±218
Epididymal WAT, mg/100 g of body weight 2605±199 3115±216 L-NAME rats had an increased food intake after 24 h of
Liver, mg/100 g of body weight 2427±55 2363±91 fasting and had a blunted response to a glucose load during
Kidneys, mg/100 g of body weight 476±16 463±13 OGTT. In 9-month-old L-NAME rats, alterations were
Heart, mg/100 g of body weight 246±11 272±6 limited and we found a slight diurnal hyperphagia, a
Interscapular BAT, mg/100 g of 144±9 113±10 ⁎ moderate hyperinsulinemia and a reduced weight of the
body weight
brown adipose tissue associated with an increased mRNA
Thymus, mg/100 g of body weight 77±7 73±6
expression of UCP1.
Lungs, mg/100 g of body weight 323±14 344±17
Adrenal glands, mg/100 g of body weight 14±1 13±1 In this model, fetal growth was reduced by a 5-days L-
Blood glucose, g/L 1.04±0.02 0.98±0.03 NAME in utero exposure that induced a 20% reduction of birth
Insulin, μU/mL 16.3±2.7 25.7±1.8 ⁎ weight and thereafter a catch-up growth during the first 10
Leptin, ng /mL 6.2±1.4 5.2±0.8 postnatal days. However, no development of a marked obesity
Triglycerides, g/L 0.91±0.04 0.68±0.09 ⁎ or diabetes was found in adult rats. We assume that the IUGR
Cholesterol, g/L 0.94±0.05 0.85±0.05 ⁎
intensity may be associated with the importance of the
Apelin, ng/mL 3.69±0.66 2.34±0.34
subsequent metabolic changes. In accordance, Ozaki et al.
Values are means±SEM, n=7–8 animals in each group. WAT, white using a model of moderate maternal dietary restriction that
adipose tissue, BAT, brown adipose tissue. resulted to a 17% reduction in the birth weight of pups did not
⁎ P<0.05.
observe any later development of obesity [7]. In contrast, a 70%
⁎⁎ P<0.01 vs controls.
reduction of caloric intake of rat mothers induced a marked
 M ET ABOL I SM CL IN I CA L A N D EX PE RI ME N TA L 6 2 ( 2 0 13 ) 44 2–4 4 5
IUGR (−35% of the birth weight) and programs a metabolic
syndrome in offspring [3].
Peripheral hormones and energetic substrates act on
feeding centers by modulating the expression and release of
hypothalamic orexigenic and anorexigenic peptides, such as
neuropeptide Y(NPY) and α-melanocyte-stimulating hor-
mone, a neuropeptide derived from proopiomelanocortin
(POMC) processing in the hypothalamus [3]. We found that
the gene expression of two food intake regulators i.e. NPY
and POMC did not differ between the two experimental
groups suggesting no disturbances in the central regulation
of food intake. We also noted a reduced relative weight of
BAT in L-NAME rats. BAT oxidizes fat and dissipates energy
produced as heat using uncoupling protein 1(UCP1), a
protein located in the inner mitochondrial membrane [8,9].
The reduction of BAT weight was correlated to a higher
UCP1 expression in L-NAME rats. We postulate that mater-
nal L-NAME treatment could alter BAT development and
that UCP1 overexpression could be a compensation to obtain
the same tissue activity than in control rats. Knowing that
UCP1 could reduce obesity and improve insulin sensitivity
after genetic or pharmacological stimulation [10], this result
could reveal an ability to adapt in order to fight against
obesity establishment.
The limitation of our experimental model is that it in-
duces some limb defects in 36% of L-NAME newborns as
previously observed [11] and a direct action of L-NAME on
fetuses cannot be eliminated. As we excluded these animals
to ensure the survival rate at the beginning of our experi-
ment, we may have selected the less affected rats, which
could explain the moderate alterations observed at 9 months
of age. We cannot exclude that more marked metabolic
alterations may be found in rats with limb defects. This
parameter could also be a limitation of using the L-NAME
rodent model to study maternal hypertension during preg-
nancy and fetuses becoming.
In conclusion, acute chronic inhibition of NO synthase
during late gestation did not seem to alter the offspring
metabolism in adulthood. While the L-NAME rat model has
strength to study maternal hypertension and fetal growth
restriction in the perinatal period, our data do not encourage
its use for works on long-term metabolic programming.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.metabol.2012.09.006.
Authors contributions
BL and LJ: conduct of the study, data collection and analysis,
data interpretation and manuscript writing. MS: data collec-
tion. ME and KC: data analysis. SL: design of the study, data
interpretation and critical reading of the manuscript. DP:
445
design and conduct of the study, data interpretation and
manuscript writing.
Funding
This work was supported by a French Ministry of Higher Edu-
cation and Research.
Acknowledgments
We would like to thank Dr. I.Fajardy for her editorial assist-
ance and A. Dive and M. Pottier for the care of animals.
Conflict of interest
The authors have nothing to declare and no conflicts of interest.
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