International Journal of Obesity (2006) 30, 739–750

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ORIGINAL ARTICLE
N-3 Fatty acids modulate antioxidant status in diabetic
rats and their macrosomic offspring
A Yessoufou1,2, N Soulaimann3, SA Merzouk3, K Moutairou4, H Ahissou5, J Prost1, AM Simonin1,
H Merzouk3, A Hichami1 and NA Khan1
1
Department of Physiology, UPRES Lipids and Nutrition, Faculty of Life Sciences, University of Burgundy, Dijon, France;
2
Faculty of Sciences and Techniques, University of Abomey-Calavi, Cotonou, Bénin; 3Department of Biology and Physics,
Faculty of Sciences, University of Tlemcen, Tlemcen, Algeria; 4Laboratory of Cell Biology and Physiology, University of
Abomey-Calavi, Cotonou, Bénin and 5Laboratory of Protein Biochemistry, University of Abomey-Calavi, Cotonou, Bénin

Objective: We investigated the role of dietary n-3 polyunsaturated fatty acids (n-3 PUFA) in the modulation of total antioxidant
status in streptozotocin (STZ)-induced diabetic rats and their macrosomic offspring.
Design: Female wistar rats, fed on control diet or n-3 PUFA diet, were rendered diabetic by administration of five mild doses of
STZ on day 5 and were killed on days 12 and 21 of gestation. The macrosomic (MAC) pups were killed at the age of 60 and 90
days.
Measurements: Lipid peroxidation was measured as the concentrations of plasma thiobarbituric acid reactive substances
(TBARS), and the total antioxidant status was determined by measuring (i) plasma oxygen radical absorbance capacity (ORAC),
(ii) plasma vitamin A, E and C concentrations, and (iii) antioxidant enzymes activities in erythrocytes. The plasma lipid
concentrations and fatty acid composition were also determined.
Results: Diabetes increased plasma triglyceride and cholesterol concentrations, whereas macrosomia was associated with
enhanced plasma cholesterol and triglyceride levels, which diminished by feeding n-3 PUFA diet. N-3 PUFA diet also reduced
increased plasma TBARS and corrected the decreased ORAC values in diabetic rats and their macrosomic offspring. EPAX diet
increased the diminished vitamin A levels in diabetic mothers and vitamin C concentrations in macrosomic pups. Also, this diet
improved the decreased erythrocyte superoxide dismutase and glutathione peroxidase activities in diabetic and macrosomic
animals.
Conclusion: Diabetes and macrosomia were associated with altered lipid metabolism, antioxidant enzyme activities and vitamin
concentrations. N-3 PUFA diet improved hyperlipidemia and restored antioxidant status in diabetic dams and MAC offspring.
International Journal of Obesity (2006) 30, 739–750. doi:10.1038/sj.ijo.0803211; published online 17 January 2006

Keywords: n-3 PUFA; diabetes; macrosomia; antioxidant status; vitamins

Introduction levels in these newborns induce oxidative stress,3 which,

Epidemiological studies have established that individuals
with diabetes have a high risk of developing atherosclerotic
vascular disease.1 One of the earliest abnormalities observed
in diabetic subjects is the involvement of oxidative stress.2
Moreover, fetuses from mothers with gestational diabetes are
at increased risk of developing fetal macrosomia, platelet
hyperaggregability and oxidative stress.3 High blood glucose
Correspondence: Professor NA Khan, Department of Physiology, Faculty of
Life Sciences, 6 boulevard Gabriel, University of Burgundy, Dijon 21000,
France.
E-mail: Naim.Khan@u-bourgogne.fr
Received 13 April 2005; revised 27 September 2005; accepted 6 October
2005; published online 17 January 2006
in turn, induces the production of highly reactive oxygen
radicals, being toxic to cells, particularly to the plasma
membranes where these radicals interact with the lipid
bilayer. Endogenous antioxidant enzymes (e.g. superoxide
dismutase, catalase, glutathione peroxidase and reductase)
and vitamins are responsible for the detoxification of
deleterious oxygen radicals.4 In diabetes as well as in
macrosomia, protein glycation and glucose autoxidation
may generate free radicals, which, in turn, catalyze lipid
peroxidation.5 Moreover, disturbances in the antioxidant
defense system in diabetes and macrosomia have been
reported as follows: alteration in antioxidant enzymes
activities,6 impaired glutathione metabolism7 and decreased
ascorbic acid levels.8 Treatment with antioxidants may
prevent or reverse abnormalities associated with diabetes
 Fatty acids in diabetes and macrosomia
A Yessoufou et al
740
and its complications. Many studies have reported that total diet). The details of the fatty acid composition of the
dietary supplements by vitamins and minerals prevent or, control diet and the EPAX diet are presented in Table 1.
at least, attenuate the organic deterioration, caused by an EPAX-7010 was tightly sealed under a stream of nitrogen to
excessive oxidative stress in diabetics.9,10 avoid lipid oxidation and kept at 41C. Diets were prepared
Contradictory results have been reported on n-3 PUFA every day and the rats consumed them ad libitum. Uneaten
consumption and lipid peroxidation, measured as thiobarbi- food was discarded; food cups and water bottles were washed
turic acid reactive substance (TBARS) levels. In fact, Wander frequently. Rats were housed individually in stainless-steel
et al.11 and Allard et al.12 have reported increased levels of cages in a room maintained at 221C with a 12-h light–dark
TBARS in human plasma after consumption of n-3 fatty acids cycle.
(EPA and DHA), whereas Nordoy et al.13 and Ando et al.14
could not find any significant differences in TBARS levels
in their studies. However, several reports have shown the Animal treatment by streptozotocin
beneficial effects of fish oils containing n-3 polyunsaturated In each diet group, a total of 25 pregnant rats were rendered
fatty acids (PUFAs) like eicosapentaenoic acid (EPA) and diabetic by intraperitoneal (i.p.) injection of streptozotocin
docosahexaenoic acid (DHA) in the protection against lipid (40 mg/kg body weight) in 0.1 M citrate buffer, pH 4.5, on the
peroxidation in rat and human beings with diabetes.15,16 fifth day of gestation. In each diet group, 10 pregnant dams
Owing to these beneficial effects of n-3 PUFA, we tested the were injected with citrate buffer alone as a control group. To
hypothesis whether EPA and DHA are implicated in preven- follow the hyperglycemia during gestation, maternal blood
tion and protection against free radical production or was collected on days 12 and day 17 of gestation, for
oxidative stress associated with lipid peroxidation in dia- determining plasma glucose concentrations, by cutting off
betes. To our knowledge, no studies have directly investi- the tip of the tail and squeezing it gently. Glycemia was
gated the role of EPA and DHA in lipid metabolism and measured in pregnant rats by One Touch II sGlucometer
oxidative stress in macrosomia. Therefore, the present study (LifeScan, Johnson and Johnson, USA). All diabetic dams
was undertaken to assess the effects of a diet supplemented included in the study had the fasting blood glucose above
with n-3 fatty acids on total antioxidant status and lipid 5.55 mM on both occasions. The success rate in obtaining
metabolism in diabetic rats and their obese offspring. these hyperglycemic dams in the current series was 60%
(15 rats out of 25 streptozotocin-treated rats). The protocol
of selection of macrosomic offspring has been well described
elsewhere.22 A total of 125 pups from the 15 streptozotocin-
Materials and methods treated dams and 91 pups from the 10 control dams were
delivered at day 21. Pups, from the streptozotocin-treated
Animals and diets dams, whose birth weights were 1.7 s.d. greater than the
Female Wistar rats, 2–3 months old and weighing 200–250 g, mean birth weight of the control pups, were classified as
were obtained from IFA-CREDO (Abresle, France). After 1
week of acclimation, animals were divided into two diet
groups. The first group of rats was fed a control diet
Table 1 Fatty acid composition of control and EPAX diets
containing vegetable oil (ISIO oil) and the other group was
fed an EPAX diet (containing EPA and DHA) for 15 days Fatty acids Control diet (mg/g) EPAX diet (mg/g)
before mating. C14 : 0 0.4 0.4
The chemical composition of the control diet was as C16 : 0 5.1 2.1
follows (g/kg dry diet): starch, 587; casein, 200; cellulose, 50; C18 : 0 3.9 1.7
sucrose, 50; mineral mix, 40; vitamin mix, 20; DL-methio- C18 : 1 18.5 9.1
C18 : 2n-6 21.3 11.2
nine, 3; vegetable oil Isio-4 (Lesieur, Neuilly-sur-Seine,
C18 : 3n-3 0.83 0.5
France), 50. Total oil comprised 5% of the diet.17–21 In the C20 : 4n-6 (AA) ND 0.9
EPAX diet, half of the vegetable oil Isio-4 was replaced by C20 : 5n-3 (EPA) ND 22.2
EPAX-7010. The composition of the mineral and the vitamin C22 : 6n-3 (DHA) ND 2.0
mix was identical to that described by Triboulot et al.17 Isio-4
Total FA 50.0 50.0
oil contained in mg/g, 47.2, 18 : 2 (n-6), 1.7, total (n-3) and
P
40.2, monounsaturated fatty acids (largely 18 : 1). EPAX- P
n-6 PUFA 21.30 12.06
7010, in the form of ethyl ester, contained approximately n-3 PUFA 0.83 24.59
(n-6)/(n-3) 25.80 0.49
85% (n-3) PUFA, that is, EPA 70%, DHA, 12%, and a-
(n-3)/(n-6) 0.04 2.04
tocopherol, 2.1–3.2%. It means that EPAX oil comprised P
SFA 9.40 4.26
P
2.5% of the diet. As the n-3 PUFA consisted of 85% of the P
PUFA 22.13 36.65
2.5% EPAX oil, the total n-3 PUFA represented only 2.1% of MUFA 18.50 9.07
PUFA/SFA 2.35 8.60
the total diet. The quantity of a-tocopherol provided to the
EPAX diet by the EPAX oil was negligible (0.05–0.08% of ND ¼ not detectable.

International Journal of Obesity


macrosomic or obese. The mean birth weight of the control
pups was 5.9070.39 g. Therefore, experimental pups with
birth weight 46.56 g were considered as macrosomic and
included in the study. These obese pups were hyperglycemic
and hyperinsulinemic. The non-macrosomic offspring of
diabetic mothers were excluded, as maternal diabetes related
to fetal macrosomia was the criterion for the selection of
our experimental population. However, these normal-sized
offspring of diabetic mothers were not hyperglycemic and
hyperinsulinemic at birth. They had normal growth rates
and did not show any significant difference from controls in
serum lipids.
At 4 weeks of age, pups of each group of rats were weaned
and fed the control or EPAX diet for 3 months with respect to
their respective maternal diet. At each experimental point,
that is, days 60 and 90, 40 macrosomic or/and 40 control rats
belonging to the control diet and EPAX diet group were
anaesthetized with pentobarbital (60 mg/kg body weight)
and killed, and blood and plasma were collected as described
below. Only the male macrosomic offspring were included in
the study.
The general guidelines for the care and use of laboratory
animals, recommended by the council of European Economic
Communities, were followed.
Blood samples
After overnight fasting, in each diet group and at each
experimental point, that is, day 12 of gestation and day 21 at
delivery, half of the pregnant dams (controls and steptozo-
tocin-treated) were anaesthetized with pentobarbital (60
mg/kg body weight) and then bled from the abdominal
aorta. Serum was obtained by low-speed centrifugation
(1000 g  20 min) and preserved with 0.26 mM EDTA and
3 mM sodium azide. Plasma was immediately used for
glucose, lipid, oxygen radical absorbance capacity (ORAC)
and vitamin determinations. After removal of plasma,
erythrocytes were washed three times with two volumes of
isotonic saline solution. Erythrocytes were lysed with cold
distilled water (1/4), stored in the refrigerator at 41C for
15 min and the cell debris was removed by centrifugation
(2000 g  15 min). Erythrocyte lysates were assayed for anti-
oxidant enzyme activities.
Determination of lipids and thiobarbituric acid-reactive
substances (TBARS)
Plasma triglyceride, phospholipid and total cholesterol
concentrations were determined using enzymatic methods,
according to the instructions furnished along with the kit
(Boehringer, Mannheim, Germany). Plasma lipids were
extracted according to the method of Bligh and Dyer,23 then
transmethylated by BF3/methanol after saponification, and
fatty acids were analyzed by gas liquid chromatography.24
Plasma free radical damage was determined by specifically
measuring TBARS in plasma.25
Fatty acids in diabetes and macrosomia
A Yessoufou et al
741
Scavenging capacity of plasma
The ORAC of plasma was measured according to Cao et al.26
A fluorescent protein b-allophycocyanin (APC) was used in
this assay.27 ORAC refers to the capacity of plasma to
scavenge free radicals, and then to delay the loss of APC
fluorescence. The reaction mixture (2 ml) in the assay
contained a final concentration of 76.8 nM APC in 75 mM
phosphate buffer pH 7.0, at 371C, 9 mM of Cu2 þ (CuSO4) and
0.3% H2O2, in the absence (blank) or the presence,
respectively, of 20 ml of Trolox 1 mM (standard) or plasma
(sample). Samples containing APC alone (blank) were
prepared to monitor the spontaneous decay of fluorescence
under experimental conditions. All readings were automati-
cally corrected for the time-dependent decay. The APC
fluorescence, at excitation 598 nm and emission 651 nm,
was measured every 5 min, at 371C, using a spectrofluorom-
eter (SFM25 Kontron Instrument, France) until the disap-
pearance of fluorescence. The ORAC value of each plasma
sample was calculated by measuring the net protection area
under the quenching curve of APC. One ORAC unit has been
assigned as the net protection area provided by 1 mM of
Trolox. The ORAC value of the samples was calculated as
ORAC ¼ (Asamples–Ablank)/(Atrolox–Ablank), A refers to the
area under the quenching curve of APC.
Determination of plasma vitamin C levels
Vitamin C levels were determined in plasma using the
method of Roe and Kuether.28 After protein precipitation
with 10% trichloroacetic acid and centrifugation, the super-
natant (500 ml) was mixed with 100 ml of DTC reagent (9 N
sulfuric acid containing 2,4-dinitrophenylhydrazine 3%,
thiourea 0.4% and copper sulfate 0.05%) and incubated at
371C for 3 h. After the addition of 750 ml of 65% (v/v) sulfuric
acid, the absorbency was recorded at 520 nm.
Determination of vitamin A and E levels in plasma
Plasma a-tocopherol (vitamin E) and retinol (vitamin A) were
determined by reverse phase HPLC.29 The stationary phase
constituted greffed silica (C18 column, HP ODS Hypersil
C18; 200 mm  4.6 mm; Lara spiral, maintenance tempera-
ture of analytical column, 351C). The mobile phase was a
mixture of methanol/water (98/2, v/v) at a flow rate of 1 ml/
min. Vitamins were extracted by hexane, dried under
nitrogen and resuspended in methanol. The extracted
vitamins were injected in the HPLC system.29 The HPLC
peaks were detected by a UV detector at 292 nm for vitamin E
and at 325 nm for vitamin A. Representative chromatograms
were obtained by injecting standard solutions.
Determinations of erythrocyte antioxidant enzyme activities
Glutathione peroxidase (GSH-Px EC 1.11.1.9) was assessed
by the method described elsewhere,30 using cumene hydro-
peroxide as substrate. One unit of glutathione peroxidase
International Journal of Obesity
 Fatty acids in diabetes and macrosomia
A Yessoufou et al
742
activity is defined as the amount of enzyme that gives a 90%
decrease in glutathione concentration per min at 1 mM
glutathione concentration. Glutathione reductase (GSSG-
Red EC 1.6.4.2) activity was determined by measuring the
rate of NADPH oxidation in the presence of oxidized
glutathione.31 The unit of enzyme activity was defined as
the amount of enzyme which oxidized 1 mmol of NADPH
per minute. Superoxide dismutase (SOD EC 1.15.1.1) activity
was measured by the NADPH oxidation procedure32 and
expressed as units of SOD per gram Hb.
Statistical analysis
Values are mean7s.d. Statistical analysis of data was carried
out using STATISTICA (version 4.1, Statsoft, Paris, France).
Data were evaluated by analysis of variance. Duncan’s
Multiple-Range test was employed for the comparison
between diabetic rats and the obese offspring with their
controls. Differences were considered significant when
Po0.05.
Results
Plasma glucose concentrations and body weight
Streptozotocin-injected pregnant rats showed an intense
hyperglycemia, whatever the diet fed (Table 2). At birth, all
the selected macrosomic pups were significantly intensely
hyperglycemic compared to the control pups. This age-
related hyperglycemia increased at days 60 and 90 (Table 3a).
Feeding the EPAX diet did not significantly influence
glycemia.
The mean birth weight of the control pups was
5.9070.39 g whereas that of the selected obese pups of the
control-diet-fed diabetic mothers was 8.0970.49 g. The
success rate in obtaining these obese pups was 64% (80 pups

Table 2 Plasma glucose concentrations (mM) in diabetic and control rats

Control diet (n ¼ 5) Diabetics, control diet (n ¼ 8) EPAX diet (n ¼ 5) Diabetics, EPAX diet (n ¼ 7)

y
Day 12 3.4470.22 16.5472.72 3.3870.27 16.3870.83y
Day 21 4.6670.22 14.4472.11y 4.5070.33 9.3872.16*,y

Values are mean7s.d. Significant differences between STZ-induced diabetic rats and their corresponding controls are as follows: yPo0.05 (diabetes effect).
Significant differences between EPAX-diet and control-diet-fed animals are as follows: *Po0.05 (dietary effect). Days 12 and 21 correspond, respectively, to the day
12 of the gestation and day 21 at delivery.

Table 3 (a) Plasma glucose concentrations (mM) in macrosomic and control offspring

Control diet (n ¼ 20) Macrosomic, control diet (n ¼ 20) EPAX diet (n ¼ 20) Macrosomic, EPAX diet (n ¼ 20)

y
Day 0 3.9470.27 9.4470.33 3.8370.27 9.1170.33y
Day 60 5.4470.5 10.7271.27y 5.4670.11 11.4471.44y
Day 90 5.3370.38 16.3871.6y 5.4770.27 16.5571.55y

(b) Body weight at birth (day 0) of the macrosomic and control offspring
Control diet EPAX diet

Offspring of control dams Offspring of diabetic dams Offspring of control dams Offspring of diabetic dams

BWp BW4
Mean BW ¼ BWp BW4 Mean BW ¼
Day 0 6.56 g 6.56 g
5.970.39 g 6.56 g 6.56 g 5.3170.25 g
n ¼ 66 n ¼ 61
n ¼ 91 n ¼ 45 n ¼ 80 n ¼ 90

(c) Evolution of the body weight (g) of the offspring

Control diet (n ¼ 20) Macrosomic, control diet (n ¼ 20) EPAX diet (n ¼ 20) Macrosomic, EPAX diet (n ¼ 20)

Day 0 5.970.39 8.0970.49y 5.3170.25 6.6370.10y,*

Day 60 25578.39 32078.01y 24574.78 28076.22y,*
Day 90 36079.01 44079.10y 340711.08 38577.09y,*

Values are mean7s.d. Significant differences between macrosomic offspring and their corresponding controls are as follows: yPo0.05. Significant differences
between EPAX-diet and control-diet-fed animals are as follows: *Po0.05 (dietary effect). Days 0, 60 and 90 correspond, respectively, to the birth, 2-month-old and
3-month-old offspring.

International Journal of Obesity


out of 125 pups of control-diet-fed diabetic dams). In
contrast, the success rate of obese pups of EPAX-diet-fed
diabetic mothers was 48% (61 pups out of 127). The
consumption of the EPAX diet diminished the incidence of
macrosomia from 64 to 48% (Table 3b). This beneficial effect
of the EPAX diet on the body weight of macrosomic offspring
was also observed at days 60 and 90 (Table 3c).
Plasma lipid concentrations
Diabetes induced an increase in total cholesterol levels in
diabetic dams compared to control animals and its levels
were significantly higher in offspring of diabetic dams
compared to control offspring (Table 4). The EPAX diet
induced a significant decrease in total cholesterol in diabetic
mothers as well as in macrosomic offspring. The offspring
(day 60) of the control dams had a high level of cholesterol
(2.60170.132 mM); however, this value remained signifi-
cantly lower than that of the macrosomic offspring
(3.78170.112 mM). Diabetes and macrosomia increased
plasma triglyceride concentrations in control-diet-fed
animals. The EPAX diet, compared to control diet, exerted
a triglyceride-lowering action in diabetic mothers as well as
Fatty acids in diabetes and macrosomia
A Yessoufou et al
743
in their obese offspring, irrespective of their age. Plasma
phospholipid levels were not significantly changed in any of
the groups.
Antioxidant enzyme activities
Diabetes mellitus diminished SOD activity in both mothers
and their offspring (Table 5). The EPAX diet induced a
significant increase in SOD activity in control and diabetic
mothers and also in their offspring.
GSSG-Red activity was not significantly affected by
diabetes mellitus at day 12 of gestation. However, from day
21 onwards, control-diet-fed diabetic dams showed lower
GSSG-Red activity when compared to their controls. GSSG-
Red activity was diminished in control-diet-fed obese off-
spring, but was increased in EPAX-diet-fed-obese offspring
compared to their corresponding controls. The EPAX diet
induced a significant increase in the activity of this enzyme
only in macrosomic pups (Table 5).
Diabetes mellitus caused a significant decrease in GSH-Px
activity in mothers and their offspring. EPAX diet induced a
significant increase in the activity of this enzyme in diabetic
mothers and their offspring (Table 5).

Table 4 Plasma lipid concentrations (mmol/l)

Dams

Control diet (n ¼ 5) Diabetics, control diet (n ¼ 8) EPAX diet (n ¼ 5) Diabetics, EPAX diet (n ¼ 7)

Total cholesterol (mmol/l)

Day 12 1.02670.092 1.81470.153y 0.99270.051 0.83170.044*
Day 21 1.54570.361 1.84570.452y 1.59170.371 1.60770.223*

Triglyceride (mmol/l)
Day 12 0.70370.052 0.99170.064y 0.64070.069 0.60270.103*
Day 21 0.98770.067 1.48270.085y 0.89970.071 0.84870.042*

Phospholipids (mmol/l)
Day 12 0.91770.116 1.60470.205 0.73370.227 1.10970.234
Day 21 2.10970.348 2.06670.503 1.75170.364 1.69770.286

Offspring

Control diet (n ¼ 20) Macrosomic, control diet (n ¼ 20) EPAX diet (n ¼ 20) Macrosomic, EPAX diet (n ¼ 20)

Total cholesterol (mmol/l)

Day 60 2.60170.132 3.78170.112y 2.66370.121 2.78970.142*
Day 90 1.67370.184 4.05670.141y 2.05670.308 2.26570.104*

Triglyceride (mmol/l)
Day 60 0.55670.054 0.79370.057y 0.53970.027 0.51170.068*
Day 90 1.07670.054 1.71270.087y 0.95770.084 0.91670.109*

Phospholipids (mmol/l)
Day 60 1.21570.154 1.02470.139 1.21770.257 1.23570.362
Day 90 0.89770.105 0.99170.106 1.20370.342 0.87970.108

Values are mean7s.d. Significant differences between STZ-induced diabetic rats or macrosomic offspring and their corresponding controls are as follows: yPo0.05
(diabetes effect). Significant differences between EPAX-diet and control-diet-fed animals are as follows: *Po0.05 (dietary effect). Days 12 and 21 correspond,
respectively, to the day 12 of the gestation and day 21 at delivery. Days 60 and 90 correspond, respectively, to 2-month-old and 3-month-old offspring.

International Journal of Obesity

 Fatty acids in diabetes and macrosomia
A Yessoufou et al
744
Table 5 Antioxidant enzyme activities in erythrocyte (U/g Hb)

Dams

Control diet (n ¼ 5) Diabetics, control diet (n ¼ 8) EPAX diet (n ¼ 5) Diabetics, EPAX diet (n ¼ 7)

SOD (U/g Hb)

Day 12 663.85797.38 455.53787.64y 874.58774.61* 613.80727.61y,*
Day 21 672.08770.29 489.16742.22y 1161.84765.16* 716.72788.73y,*

GSSG-Red (U/g Hb)

Day 12 41.2474.02 36.2774.79 39.6773.24 35.7171.09
Day 21 72.1774.56 56.5473.89y 69.4777.45 58.8678.95

GSH-Px (U/g Hb)

Day 12 92.6979.51 63.2273.51y 107.8177.52 75.0173.20y,*
Day 21 100.4579.34 73.5671.87y 112.8472.29 98.5778.92y,*

Offspring

Control Diet (n ¼ 20) Macrosomic, control diet (n ¼ 20) EPAX diet (n ¼ 20) Macrosomic, EPAX diet (n ¼ 20)

SOD (U/g Hb)

Day 60 893.74790.01 580.86768.76y 1144.91778.88* 802.35754.39y,*
Day 90 955.76766.05 394.81739.55y 1050.327 36.75* 806.09754.56y,*

GSSG-Red (U/g Hb)

Day 60 62.9377.06 34.7478.52y 58.3877.31 67.83711.71y,*
Day 90 67.6379.03 28.6472.82y 62.6372.34 84.5478.31y,*

GSH-Px (U/g Hb)

Day 60 89.9475.49 59.9377.39y 99.2779.13 71.4873.68y,*
Day 90 98.9179.62 61.4572.41y 114.8375.73 97.6174.69y,*

Values are mean7s.d. Significant differences between STZ-induced diabetic rats or macrosomic offspring and their corresponding controls are as follows: yPo0.05
(diabetes effect). Significant differences between EPAX-diet and control-diet-fed animals are as follows: *Po0.05 (dietary effect). Days 12 and 21 correspond,
respectively, to the day 12 of the gestation and day 21 at delivery. Days 60 and 90 correspond, respectively, to 2-month-old and 3-month-old offspring. SOD,
superoxide dismutase; GSH-Px, glutathione peroxidase; GSSG-Red, glutathione reductase.

Total fatty acid compositions of plasma lipids Plasma TBARS

In dams at day 21. In the plasma lipids of pregnant rats, no
significant change was observed in the percentages of
saturated fatty acids (SFA) between diabetics and controls;
however, these percentages were significantly lower in the
EPAX-diet-fed group than the control-diet-fed group. In the
control-diet-fed group, the proportions of linoleic acid (LA,
C18 : 2n-6) significantly increased in diabetic animals com-
pared to controls, whereas those of arachidonic acid (AA)
decreased. Feeding the EPAX diet induced increased levels of
EPA and DHA, whereas the levels of AA decreased in the non-
diabetic group and remained stable in the diabetic group
(Table 6).
In offspring at days 60 and 90. In the plasma lipids of
offspring, there was no change in the proportions of SFA
(16 : 0; 18 : 0) between obese and control offspring, irrespec-
tive of the diet. In the control-diet group, macrosomia was
associated with an increased level of linoleic acid (18 : 2n-6)
and a decreased level of AA (20 : 4n-6). EPAX diet consump-
tion induced an increased level of EPA and DHA, and
corrected the changes induced by diabetes in LA and AA
proportions, particularly at day 90 (Table 7).
Diabetic rats and their offspring exhibited high plasma
TBARS compared to control rats, irrespective of their age
(Figure 1). Feeding EPAX diet significantly reduced the
marked increases in TBARS induced by diabetes and macro-
somia.
Plasma total antioxidant status
Compared with controls, ORAC was significantly low in
diabetic rats and their macrosomic offspring (Figure 2). The
EPAX diet induced a significant increase in ORAC values in
diabetic dams and macrosomic offspring, irrespective of their
age.
Plasma vitamin C concentrations
Plasma vitamin C levels were not altered in diabetic mothers
(results not shown). However, macrosomic pups, fed on the
control diet, had low levels of vitamin C compared with
controls. Dietary EPAX enhanced and improved plasma
vitamin C concentrations in this group up to the level of
controls (Figure 3a).

International Journal of Obesity

 Fatty acids in diabetes and macrosomia
A Yessoufou et al
745
Table 6 Percent total fatty acid composition of plasma

Dams at day21

Fatty acids Control diet (n ¼ 5) Diabetics, control diet (n ¼ 8) EPAX diet (n ¼ 5) Diabetics, EPAX diet (n ¼ 7)

C16 : 0 28.470.7 28.271.2 19.273.3* 19.571.3*

C18 : 0 18.871.2 16.471.5 13.671.4* 14.872.5
C18 : 1 16.771.6 18.272.9 11.971.5* 12.571.8*

C18 : 2n-6 12.270.6 21.571.5y 11.670.6 14.870.7y,*

C20 : 4n-6 (AA) 21.870.1 11.070.2y 12.272.1* 10.371.4

C20 : 5n-3 (EPA) 0.770.5 2.271.2 18.171.3* 16.971.7*

C22 : 6n-3(DHA) 1.371.0 2.471.4 13.470.6* 11.272.8*

Offspring
At day 60 Control diet (n ¼ 20) Macrosomic, control diet (n ¼ 20) EPAX diet (n ¼ 20) Macrosomic, EPAX diet (n ¼ 20)

y
C18 : 2n-6 15.872.1 20.072.1 9.970.4* 13.171.0y,*
C20 : 4n-6 (AA) 23.373.0 16.773.2y 11.172.1* 7.970.2y,*

C20 : 5n-3 (EPA) 1.070.2 1.370.4 13.371.0* 14.171.6*

C22 : 6n-3 (DHA) 1.270.3 1.970.4 11.771.0* 7.070.7y,*

At day 90
C18 : 1 19.771.5 16.472.5 13.270.2* 13.772.1*

y
C18 : 2n-6 17.370.6 23.672.1 10.470.4* 11.570.5*
C20 : 4n-6 (AA) 22.970.7 19.870.2y 11.070.5* 11.771.6*

C20 : 5n-3 (EPA) 0.770.3 0.770.3 15.871.0* 10.571.8y,*

C22 : 6n-3 (DHA) 2.470.7 2.070.4 9.970.3* 8.971.8*

Values are mean7s.d. Only main fatty acids with significant differences have been shown. Significant differences between STZ-induced diabetic rats or macrosomic
offspring and their corresponding controls are as follows: yPo0.05 (diabetes effect). Significant differences between EPAX-diet and control-diet-fed animals are as
follows: *Po0.05 (dietary effect). Day 21 corresponds to the day 21 at delivery. Days 60 and 90 correspond respectively to 2-month-old and 3-month-old offspring.

Table 7 Effects of n-3 fatty acids on antioxidant status as reported by various investigators

Antioxidant status Species n-3 PUFA level in the diet References

Decreased Diabetic rats 10% of diet (considered as excessive) Cho and Coi54
Decreased Healthy humans EPA: 2.5 g/day; DHA: 1.8 g/day Wander and S-H Du11
Decreased Healthy humans 6.26 g/day for 6 weeks Allard et al.12
Decreased Patients with myocardial infraction 850–882 mg/day (EPA+DHA) for 1 year Grundt et al.53
Decreased Diabetic rats Fish oil Yilmaz et al.50
Unchanged Healthy humans 4 g/day (n-3) PUFA for 5 weeks Hansen et al.55
Unchanged Rats n-3 fatty acid-rich diet (fish oil) Ando et al.14
Unchanged Hyperlipidaemic Patients 4 g/day (DHA or EPA) Nordoy et al.13
Improvement Diabetic humans EPA: 1.08 g/day; DHA: 0.72 g/day Kesavulu et al.51
Improvement Diabetic rats 2.1% of diet Present study

Plasma vitamin A and E concentrations Discussion

Plasma vitamin A concentrations, diminished in diabetic
mothers, were normalized by feeding EPAX diet, irrespective
of the gestational age (Figure 3b). Compared to controls,
macrosomic pups fed the control diet exhibited lowered
plasma vitamin A concentrations at day 90 only. In this
group, feeding EPAX diet enhanced and restored its con-
centrations (Figure 3c). The levels of vitamin E were
unaltered in diabetic and macrosomic animals. Similarly,
feeding EPAX diet failed to influence plasmatic vitamin E
levels (results not shown).
In the present study, we investigated the role of n-3 PUFA
(essentially EPA, DHA) on lipid metabolism and total
antioxidant status in streptozotocin (STZ)-induced diabetic
rats and in their macrosomic offspring. The administration
of streptozotocin (STZ) to mice33–35 and rats36 represents a
good model of diabetes development for several reasons: (i)
islet lesions in this experimental model resemble those of
human insulitis,35 (ii) the animals used are normal and do
not have underlying immune abnormalities like BB rat37 and

International Journal of Obesity

 Fatty acids in diabetes and macrosomia
A Yessoufou et al
746
a Dams a Dams Control diet
Control diet Diabetics, control diet
60
Diabetics, control diet 3 EPAX diet
§§
EPAX diet Diabetics, EPAX diet
TBARS (µmoles/L plasma)

Orac, arbitrary units

50 2.5 **
Diabetics, EPAX diet **
40 2
§§
1.5
30 ** * §§
1 §§
20 0.5
0
10
day12 day21

0 b Control diet
Offspring
day12 day21 Macrosomic, control diet
EPAX diet
b Offspring Macrosomic, EPAX diet
Control diet 3

Orac, arbitrary units

140 Macrosomic, control diet 2.5
**§
§§ EPAX diet 2 *§
120 §§
TBARS (µmoles/L plasma)

Macrosomic, EPAX diet 1.5 §§

100 §§
**§
80
**§
60
40
20
0
day60 day90
Figure 1 Plasma TBARS concentrations in diabetic and control rats (a) and
their offspring (b). Plasma TBARS concentrations were determined as
described in the Materials and methods section. Values are mean7s.d.
Significant differences between STZ-induced diabetic rats or macrosomic
offspring and their corresponding controls are as follows: yPo0.05 and
yy
Po0.01. Significant differences between EPAX-diet and control-diet-fed
animals are as follows: *Po0.05 and **Po0.01. Days 12 and 21 correspond,
respectively, to day 12 of gestation and day 21 at delivery. Days 60 and 90
correspond, respectively, to 2-month-old and 3-month-old offspring.
NOD mice,38 and (iii) the onset of diabetes is controlled. As
far as the model of gestational diabetes and their macro-
somic offspring is concerned, this model is well characterized
and established in our laboratory by administrating STZ
to Wistar female rats.16,22,24,39 Hence, we have selected only
male macrosomic offspring for the study. Indeed, sex
hormones have been associated with the prevalence, the
susceptibility and the severity of autoimmune diseases.40
Moreover, immune responses between male and female
rats are not identical,41 and the dietary EPA/DHA highly
influences immune response.20 Had we chosen the male and
female progenies, our results would have overlapped.
We observed that macrosomic offspring of diabetic dams
were hyperglycemic at birth and had accelerated growth
compared to offspring of control rats – independently of
maternal diet.22 Feeding the n-3 PUFA diet significantly
reduced the incidence of gestational diabetes on macrosomia
1
0.5
0
day60 day90
Figure 2 Plasma ORAC values in diabetic and control rats (a) and their
offspring (b). Plasma ORAC values were determined as described in the
Materials and methods section. Values are mean7s.d. Significant differences
between STZ-induced diabetic rats or macrosomic offspring and their
corresponding controls are as follows: yPo0.05 and yyPo0.01. Significant
differences between EPAX-diet and control-diet-fed animals are as follows:
*
Po0.05 and **Po0.01. Days 12 and 21 correspond, respectively, to day 12
of gestation and day 21 at delivery. Days 60 and 90 correspond, respectively,
to 2-month-old and 3-month-old offspring.
by decreasing the rate of macrosomic pups at birth from 64
to 48%. However, n-3 PUFA diet had no effect on
hyperglycemia of macrosomic offspring. Furthermore, dia-
betes in pregnant rats was associated with hypertriglycer-
idemia as reported by several investigators.42,43 Maternal
diabetes mellitus also induced hypercholesterolemia and
hypertriglyceridemia in obese offspring, and this observation
corroborated our previous studies.22,24,39 In the present
study, we observed a high level of cholesterol in the offspring
of the control group at day 60; however, this level remained
significantly lower than that of macrosomic offspring. In
fact, a significant age-related change in serum cholesterol
concentration has been evoked,44 and this can be explained
by the existence of a compensatory mechanism for main-
tenance of hepatic cholesterol homeostasis with age.45 It has
been shown that, in diabetic rats, high levels of triglyceride
in maternal circulation may create a steep concentration
gradient across the placenta, which accelerates their trans-
port and deposition in fetal tissues.43 This hypertriglycer-
idemia has been shown to persist with age and has been
linked to the development of insulin sensitivity and
hyperlipogenesis.46 Feeding EPAX diet significantly

International Journal of Obesity

 Fatty acids in diabetes and macrosomia
A Yessoufou et al
747
Control diet significant decrease was observed in TBARS after feeding
Diabetics or Macrosomic, control diet n-3 PUFA to the diabetic rats and their obese offspring. These
EPAX diet findings are substantiated by several investigators48,49 who
Diabetics or Macrosomic, EPAX diet have reported a decreased production of TBARS in human
subjects treated with DHA and EPA. In fact, endogenous
a Offspring
antioxidant enzymes and vitamins are responsible for the
35
Vitamin C, µmol/L

30 detoxification of deleterious oxygen radicals.4 Diabetes, in

** **
25 our study, leads to a significant decrease in the plasma total
20 §§ §§ antioxidant status as measured by diminished ORAC in rats
15 fed on the control diet. Several authors, including McLennan
10 et al.7 and Young et al.8have also demonstrated diminished
5 antioxidant enzyme activities and vitamin levels in STZ-
0
induced diabetic rats. Similarly, macrosomic pups exhibited
day60 day90
diminished plasma ORAC, vitamin C concentrations, SOD
b 18 Dams and GSH-Px activities. Similar results have been obtained by
16 Dincer et al.6 who have shown a decrease in these enzyme
Vitamin A, µmol/L

** activities in the liver and lung of neonate STZ-induced

14
12 §§ diabetic rats. In the present study, we have observed that
10
8 ** feeding EPAX diet reduces the oxidative stress, induced by
6 diabetes and macrosomia. The EPAX diet also corrected
4 §§ plasma ORAC levels in diabetic rats and macrosomic pups.
2
This could be related to increased plasma vitamin A levels
0
day12 day21 and improved erythrocyte antioxidant enzyme activities.
Our results are consistent with other reports on the
c
Offspring reduction of lipid peroxidation and an improvement in the
20
Vitamin A, µmol/L

18 * activities of antioxidant enzymes with n-3 PUFA in human

16 § beings and animals with experimental diabetes.15,21,50–52
14
12
10
8 molecule. In the EPAX diet, its quantity was very negligible
6 (0.05–0.08%). The antioxidant properties cannot be solely
4
2 contributed by alpha-tocopherol as it was present in both
0
day60 day90
Figure 3 Plasma vitamin C and vitamin A concentrations in diabetic and
control rats and their offspring. (a) Vitamin C concentrations in control and
macrosomic offspring. Plasma vitamin C concentrations were determined as
described in the Materials and methods section. Values are mean7s.d.
Significant differences between macrosomic offspring and their corresponding
controls are as follows: yyPo0.01. Significant differences between EPAX-diet
and control-diet-fed animals are as follows: **Po0.01. Days 60 and 90
correspond, respectively, to 2-month-old and 3-month-old offspring. (b and
c) Plasma vitamin A concentrations in diabetic and control rats and their
offspring. Plasma vitamin A concentrations were determined as described in
the Materials and methods section. Values are mean7s.d. Significant
differences between STZ-induced diabetic rats or macrosomic offspring and
their corresponding controls are as follows: yPo0.05 and yyPo0.01. Significant
differences between EPAX-diet and control-diet-fed animals are as follows:
*
Po0.05 and **Po0.01. Days 12 and 21 correspond, respectively, to day 12
of gestation and day 21 at delivery. Days 60 and 90 correspond, respectively,
to 2-month-old and 3-month-old offspring.
decreased triglyceride and cholesterol concentrations in
diabetic rats. EPAX diet attenuated hyperlipidemia, a long-
term complication of macrosomia, in offspring of diabetic
dams. This observation is in accordance with previous
findings, which have shown that n-3 PUFA-enriched diets
decrease both plasma triglyceride and cholesterol levels.47
Diabetic rats and their macrosomic offspring exhibited
a significant increase in plasma TBARS. In our study, a
Alpha-tocopherol is known as a liposoluble antioxidant
standard and experimental diets. However, the antioxidant
properties might have principally been contributed by EPA
or DHA as these fatty acids were abundantly present in the
experimental diet. EPA and DHA belong to the same family
of fatty acids as they are derived from the same precursor. As
the EPAX oil is rich in EPA, we can assume that this fatty acid
may largely contribute to the antioxidant properties in our
study. However, the general notion is that n-3 PUFA might
deteriorate antioxidant capacity. Nonetheless, no consensus
has been reached on this subject as shown in Table 7.
Excess intake of n-3 PUFA may reduce antioxidant
status,11,12,50,53,54 thus enhancing susceptibility to oxidative
damage. Other investigators13,14,55could not find any change
in antioxidant status in humans and rats treated with n-3
fatty acid-rich diet. On the contrary, Kesavulu et al.51 have
demonstrated that the treatment of diabetic patients with
n-3 fatty acids improved their antioxidant status. In our study,
a significant improvement was also observed in antioxidant
status in n-3 PUFA-fed diabetic rats and their obese offspring.
It has been shown that dietary fish oil alters the composition
of plasma membrane phospholipids by increasing EPA and
DHA contents at the expense of AA levels.14 This substitu-
tion may diminish or counterbalance the negative effects of
AA (n-6 PUFA) on antioxidant status. The balance between n-
3 and n-6 fatty acids may markedly affect cell metabolism.

International Journal of Obesity

 Fatty acids in diabetes and macrosomia
A Yessoufou et al
748
Indeed, essential fatty acids pattern influences the physical
properties of cell membranes (fluidity and permeability), the
activity of membrane receptors, enzymes and ion channels
and cell response to various stimuli through the production
of secondary messengers. Hence, the beneficial effect of
EPAX diet on antioxidant status will involve DHA and EPA.
In addition, EPA is known to give rise to eicosanoids of n-3
series, which exert opposite effects to those of n-6 series,
derived from AA. Besides, EPA may also be converted into
DHA, which along with dietary DHA, may further contribute
to the beneficial effects. It has been recently shown that DHA
may also give rise to some recently discovered derivatives
like docosatrienes or resolvins, which also exert beneficial
effects.56 Besides, these fatty acids have been shown to
modulate cell signaling mechanisms via their incorporation
into the plasma membrane phospholipids.57 Although the
exact mechanism by which EPA/DHA exerted antioxidant
action is not well understood, Das et al.58 have suggested in
their study that EPA/DHA supplementation inhibited free
radical generation and suppressed lipid peroxidation and NO
synthesis in patients with nephritic syndrome. This finding
suggests that EPA or DHA may be involved in scavenging of
free radicals and NO. Similarly, Yazu et al.59 have reported
that in aqueous, micellar dispersions composed of methyl
esters of EPA or linoleate, the oxidizability of the methyl
ester of EPA was lower than that of methyl linoleate. The EPA
micelle had X2 molecules of oxygen in the peroxyl radical,
whereas the linoleate micelle had only 1. These authors
argued that the EPA was more polar than linoleate, and
the polar radicals might migrate from the lipophilic core of
the micelle to the polar surface. Owing to this migration, an
environment was created that favored the termination and
reduced the propagation of oxidation reactions.11 This is one
of the mechanisms which may explain that EPA possesses
antioxidant properties and it behaves as a peroxyl and free
radical scavenger; however, more studies are needed to
elucidate the mechanism of action of DHA.
An increase in the levels of plasma fatty acids, without
dietary supplementation during normal pregnancy, has been
reported.60 Dunstan et al.61 have observed that supplement-
ing the diet with fish oil (EPA and DHA), during pregnancy,
resulted in an increase in EPA and DHA in phospholipids of
maternal peripheral blood at 30 and 37 weeks of gestation,
and remained elevated at 6 weeks postpartum. However, in
the present study, we have observed that the dietary
supplementation with EPA and DHA prevented hyperlipide-
mia in diabetic pregnant mothers and macrosomic offspring.
Our findings are in accordance with several studies which
have reported that dietary n-3 PUFA might counteract
maternal and fetal hypertriglyceridemia and might decrease
weight gain associated with macrosomia.16,62
In our study, diabetes causes a major decline in plasma AA
and an increase in LA concentrations in dams and their
macrosomic offspring, and this may be due to an impaired
activity of D5 and D6 desaturases.63 The complications
associated with diabetes, such as increased oxidative stress
International Journal of Obesity
and hyperglycemia, could be related to this intense decline
of plasma AA concentrations, as some studies have shown
that AA may have a critical role in maintaining the
appropriate mass and function of islet beta-cells by influen-
cing rates of cell proliferation and insulin secretion.64
The novel finding of our study is that a moderate n-3 PUFA
diet improves antioxidant status in rats during diabetic
pregnancy and their adult macrosomic offspring. In fact,
macrosomia is a growing health problem all over the world.
One of the causes of macrosomia is diabetic pregnancy. No
study is available on the improvement of antioxidant status
by moderate n-3 PUFA in these two pathologies. Our study
adds another factor in the etiopathology of macrosomia and
diabetic pregnancy. As feeding fish oil showed a beneficial
effect by increasing the antioxidant defense system, we can
allude that an adequate dose of fish oil may be recom-
mended in foods to reduce the incidence and complications
associated with macrosomia of diabetic pregnancy.
Acknowledgements
We thank the Office of Scholarship Programme of IDB which
granted a scholarship to A. Yessoufou. We also thank the
French Embassy at Cotonou, Benin and the French Foreign
Office (CMEP), Burgundy Region which sanctioned the
contingent grants for this work.
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