Prostaglandins & other Lipid Mediators 78 (2005) 231–248

Long-term intake of edible oils benefits blood

pressure and myocardial structure in spontaneously
hypertensive rat (SHR) and streptozotocin
diabetic SHR
Fernanda Jurema Medeiros a,c , Cheila Gonçalves Mothé b ,
Márcia Barbosa Aguila a , Carlos Alberto Mandarim-de-Lacerda a,∗
aUniversidade do Estado do Rio de Janeiro (UERJ), Centro Biomédico, Instituto de Biologia, Laboratório de
Morfometria e Morfologia Cardiovascular. Av. 28 de Setembro, 87 (fds). Rio de Janeiro, RJ 20551-030, Brazil
b Department of Organic Process, School of Chemistry, Federal University of Rio de Janeiro (UFRJ), Brazil
c Department of Applied Nutrition, Federal University of State of Rio de Janeiro (UNI-RIO), Brazil

Received 14 July 2005; received in revised form 19 August 2005; accepted 1 September 2005
Available online 30 September 2005

Abstract

The beneficial effects of edible oils long-term supplementation in blood pressure (BP) and cardiac
structure were investigated in spontaneously hypertensive rat (SHR) and streptozotocin diabetic (Db)
SHR (45 mg/rat i.p.). Twenty-five 12-week old male SHR were divided into four SHR-Db groups
and one SHR group, SHR-Db groups each receiving, respectively, olive oil, palm oil and fish oil, and
another SHR-Db group with placebo by gavage on a daily basis for 6 weeks. Myocardial structures
were analyzed through light microscopy and stereology. In SHR-Db, the BP and the myocardium were
significantly altered by oil supplementation. The BP, the interstitial fibrosis and cardiomyocyte size
showed a significant decrease in treated SHR-Db than in SHR or untreated SHR-Db. The myocardial
microvasculature and number of cardiomyocytes were higher in all treated groups, especially in fish
oil group. Long-term edible oil supplementation showed beneficial effects decreasing BP levels and
offsetting adverse myocardial remodeling in diabetic SHR.
© 2005 Elsevier Inc. All rights reserved.

Keywords: Diabetes; Hypertension; Cardiac remondeling; Fish oil; Palm oil; Olive oil; Stereology; Rat

∗ Corresponding author. Tel.: +55 21 2587 6416; fax: +55 21 2587 6416.
E-mail address: mandarim@uerj.br (C.A. Mandarim-de-Lacerda).

1098-8823/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.prostaglandins.2005.09.001
232 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248

1. Introduction

The combination of hypertension and diabetes increases the cardiovascular risk with dra-
matic complications such as atherosclerosis, heart and kidney diseases, cerebral stroke and
metabolic syndrome [1,2] and these complications are related to lipid disorders, in which
polyunsaturated fatty acids (PUFA) metabolism is particularly altered [3]. Therefore, nutri-
tional intervention is helpful in the long-term treatment of these alterations in association
with current pharmacological treatments.
Under physiological conditions, most of the energy for myocardial contraction is derived
from fatty acid oxidation [4]. PUFA are known to play important roles in various biolog-
ical functions, in particular as membranes components, precursors of second messengers
and regulators of key genes of lipid metabolism [5]. Genetic hypertension alters PUFA
biosynthesis and fatty acid composition of hepatic membranes. Indeed, and inhibition of
desaturase activities and an increase of monounsaturated fatty acids (MUFA) in microsomal
membranes, where desaturation occurs, have been observed in spontaneously hypertensive
rats (SHRs) [6,7] and diabetes can also alter PUFA metabolism and eicosanoid biosynthesis
[8]. Desaturase activities are also modified in streptozotocin-induced type 1 diabetes [9].
The consumption of n-3 PUFA rich diet has been shown to successfully reduce blood
pressure in SHR [10], decrease the incidence of coronary heart disease, and suppress cardiac
arrhythmia [11–13]. Therefore, we studied the effects of genetic hypertension combined
with streptozotocin administration on blood pressure and cardiac structure of SHR fed
different edible oils (olive oil, monounsaturated lipid source; palm oil, saturated lipid source
and fish oil, polyunsaturated n-3 lipid source).

2. Methods and materials

2.1. Animals and diet

Animal experimentation procedures were approved by the Ethic committee on Exper-

imental Animals of the State University of Rio de Janeiro, Rio de Janeiro, Brazil. All
procedures were carried out in accordance with the conventional guidelines for experimen-
tation with animals (NIH Publication No. 85-23, revised 1996).
Twenty-five 12-week-old male SHR from colonies maintained at the State University
of Rio de Janeiro were housed under controlled temperature (21 ± 2 ◦ C) and humidity
(60 ± 10%) with 12 h:12 h day/night cycle (artificial lights, 19:00–07:00 h) and a air replace-
ment cycle of 15 min/h. Animals had free access to food (commercial standard pellets;
Nuvilab, Brazil) and water. They were randomly divided into five groups of five animals
each.
Experimental type 1 diabetes mellitus was induced in four groups of SHR by a single
streptozotocin (Sigma Chemical Co., St. Louis, USA) injection (45 mg/kg intraperitonial,
dissolved in 50 mmol/l sodium citrate buffer, pH 4.5). SHR were fasted overnight before
streptozotocin administration and the SHR control group received citrate buffer alone.
Before the streptozotocin injection and 24 h later, blood glucose was measured using a
glucometer Accu-Chek Advantage (Roche, Sao Paulo, Brazil) to confirm the development
 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248 233

of diabetes mellitus. Only animals showing weight loss (because of uncontrolled diabetes),
severe hyperglycemia (fasting blood glucose levels greater than 300 mg/dl) were considered
to have developed type 1 diabetes mellitus. SHR diabetic (SHR-Db) and SHR control were
maintained for 6 additional weeks on the corresponding edible oils treatment. Metabolic
control was evaluated based on plasma glucose and animal weight at the end of the experi-
ment.
The diabetic SHR were randomly assigned on a daily basis different edible oils (1.5 g/kg
body mass/per day) or placebo (water) during 6 weeks via a stomach tube (gavage, 8:00
a.m.). The groups were divided as follows: (a) olive oil (source of n-9 monounsaturated
fatty acids, oleic acid obtained from extra-virgin olive oil, European source) (SHR-Db-
olive group), (b) palm oil (predominantly the saturated fat, palmitic acid; obtained from the
Malaysian Palm Oil Promotion Council) (SHR-Db-palm group), (c) fish oil (source of the
n-3 polyunsaturated fatty acids EPA and DHA; Sigma Chemical Co., St. Louis, USA, Batch
# 035K0184) (SHR-Db-fish group) and (d) water to both diabetic control group (SHR-Db-
group) and (e) SHR control group (SHR group). During the study, all groups had standard
rat chow available ad libitum in addition to the fat supplement, and had free access to
water.
The fatty acid composition of the palm oil used in this study was supplied by manufac-
turers (palm oil: www.mpopc.org.my), and the fatty acid composition of the olive oil was
supplied by Geygy Scientific Tables [14] based on the European source (Spain, France,
Italy). The oils were maintained in appropriated opaque recipient at room temperature. Fish
oil is considered to be standard refined Menhaden oil. Whole fish was used to prepare this
product as the fish are very small and difficult to dissect in any way. It was considered to be
refined since the crude product was treated in the following manner: (a) It was reacted in a
saponification reaction with sodium hydroxide to neutralize any free fatty acids and obtain
the sodium salt, (b) the product was bleached, (c) acid activated earth was added to the oil
to remove color bodies, metals and oxidation products, and (d) the product was then chilled
to form the solid waxy material (there are two fractions at room temperature), which was
then filtered out leaving the oil. No antioxidant or other stabilizers are added to the oil. The
composition of the supplements is given in Table 1. The fatty acid composition of the fish
oil used in this study was determined by high resolution gaseous chromatography (Hewlett
Packard, 5890 GC-FID System, Houston, TX, USA) [15].

2.2. Blood pressure (BP) and body mass measurement

Systolic blood pressure of conscious rats was measured every week (since the day before
the streptozotocin administration until the end of the experiment) by a tail-cuff plethysmo-
graphic non-invasive method (Letica LE 5100, Panlab, Barcelona, Spain). The body mass
and the naso-anal length were also weekly measured throughout the period of the experi-
ment.

2.3. Euthanasia

The night before the euthanasia, animals were kept without food in metabolic cages
to collect 24 h urine. Fifteen minutes before euthanasia, they were deeply anaesthetized
234 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248

Table 1
Fatty acid composition of different dietary lipid supplements (g fatty acid per 100 g total fatty acids)
Fatty acid Olive oil Palm oil Fish oil
(European source) (www.mpopc.org.my) (menhaden)
12:0 – – 0.1
13:0 – – 0.1
14:0 – 1.0 8.3
14:1 – – –
15:0 – – 0.7
16:0 9.1 41.0 17.1
16:1 <3 <3 10.9
17:0 – – 0.5
17:1 – – 1.4
18:0 <3 4.5 3.1
18:1 trans – – 1.6
18:1 (
9) 81.6 41.0 7.0
18:1 (
11) – – 3.2
18:2 cis/trans – – 1.0
18:2 5.3 9.5 0.3
18:3 trans – – 0.4
18:3 <3 <3 1.5
18:4 – – 2.8
19:0 – – 0.1
20:0 – <3 0.2
20:4 – – 1.0
20:5 – – 12.9
22:5 – – 2.6
22:6 – – 12.0
24:1 – – 0.3
SFA 11.6 46.9 30.2
MUFA 82.4 41.3 24.3
PUFA 6.0 9.9 34.6
n-3 PUFA <3 <3 26.8
n-6 PUFA 5.3 9.5 2.3
Abbreviations: FA—fatty acids, MUFA—monounsaturated fatty acids, PUFA—polyunsaturated fatty acids,
SFA—saturated fatty acids.

(intraperitoneal sodium pentobarbitone) and then thorax was opened, right atrium wall
was cut, and the vascular system was perfused with constant pressure (90 mmHg)
through a catheter put into the left ventricle (LV) with fixative (freshly prepared
4%, w/v formaldehyde in 0.1 M phosphate buffer pH 7.2) [16] until animal body
rigidity.

2.4. Stereology

The heart was removed; the atria were separated from the ventricles and the right ventricle
from the LV. The heart and the LV volumes (including the interventricular septum) were
measured by liquid displacement (Scherle’s method) [17], in which the organ volume is
recorded by weight (W) (as the isotonic saline specific gravity (σ) is of 1.0048, the respective
 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248 235

volumes were obtained by V[organ] = W[organ]/σ or simply V (cm3 ) ∼ = W (g) [18]. The LV
mass index was determined as the LV mass/body mass ratio.
The LV fragments were fixed by immersion for 48 h in the same fixative used in the
perfusion and then embedded in Paraplast plus (Sigma Chemical Co., St. Louis, USA),
sectioned 6 ␮m thick, and sections were stained with hematoxylin-eosin and trichrome
methods (Masson and picro sirius red stains). The myocardium was analyzed consider-
ing the cardiomyocytes (cmy) and the cardiac interstitium (intramyocardial arteries, ima,
and the connective tissue, ct). The volume density was estimated for cmy, ima and ct:
(Vv[structure] = Pp [structure]/PT ) (Pp is the number of points that hit the structure; PT is
the total test-points inside the test-system). The ratio between ima/cmy was used to study
the amount of myocardial vascularization and was estimated as the ratio Vv[ima]/Vv[cmy].
Density per area was estimated for cmy and ima, QA [structure] = N[structure]/AT (1/mm2 , N
is the number of profiles counted in the test-area, the frame AT , considering the forbidden line
or its extensions [19]). Length density was estimated for ima, Lv[ima] = 2QA [ima] mm/mm3
and the mean cross-sectional area was estimated for cmy, A[cmy] = Vv[cmy]/2QA [cmy]
(␮m2 ) [20].
The disector method used in the present study to estimate the number of cmy is a
three-dimensional probe that samples structures proportional to their number without
regard to the size or shape of the structures [21]. In a disector, two sections are used
to create a sampling volume with an upper, reference section, containing a test frame.
Sections were viewed with a 100× planachromatic immersion oil objective on a Leica
DMRBE microscope (NA = 1.25) to identify cmy nuclei. For each frame, the thickness
of the section was measured by focusing on the upper and lower section surfaces ver-
ified objectively by employing an auto-focus device and read-out (the microscope was
equipped with a z-axis motorized focus controller microcator with a resolution of 0.1 ␮m).
Light microscopy was performed using a microscope Leica DMRBE (Wetzlar, Germany),
a Kappa videocamera (Gleichen, Germany) and a Sony Trinitron monitor (Pencoed,
UK).
The numerical density (Nv) of cmy (number of cmy per mm3 ) was determined
from 10 random disector pairs for each rat. This sampling design was based on a
pilot experiment to determine the inter-animal variability. Estimates of relative variance
(=variance/mean2 = [coefficient of variation]2 ) of around 10% was considered acceptable
[22]. For reasons of efficiency, we analyzed the nuclei of the cmy (one nucleus represented
one cardiomyocyte), centers of nuclei were measured, rather than tops of nuclei. The center
of nuclei was defined by focusing on the clear nuclear edge and the most clearly defined
nuclear chromatin to prevent an asymmetric distribution profile. The thickness of the dis-
ector (t) was 3 ␮m that represented 1/4 to 1/3 of the height of the cmy nuclei, which in
practice is the more important constraint on section thickness [23].

Q−
cmyn
Nv [cmyn] =
tAT

An example of the disector method used in the present study is showed in Fig. 1. The total
number of LV cmy (N) was calculated by multiplying the Nv by the LV volume, previously
estimated.
236 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248

Fig. 1. The photomicrographs of the myocardium to demonstrate the disector method used in this study (calibration
bar = 10 ␮m, stain H-E). Both planes inferior (A) and superior (B) were taken from the same microscopical field with
a distance of 3 ␮m. A frame of known area incorporating the “forbidden lines” (thick black line) was superimposed
on the fields to delineate the observational area (all nuclei crossed by these lines were not considered—arrows). In
the inferior plane, only one sharp nucleus was counted while in the superior plane four sharp nuclei were counted.

2.5. Data analysis

The differences in biochemical and biometric parameters among the groups were tested
by one-way analysis of variance and the Newman–Keuls post-hoc test. Differences in
stereology were tested by non-parametric analysis of variance of Kruskal–Wallis. When dif-
ferences were significant, the Mann–Whitney test was used to test the parameters between
two groups. In all cases the level of significance was set at p < 0.05 [24].

3. Results

No diarrhea or any physical changes of the animals were attributed to lipid supplemen-
tation in this work. However, the induction of type 1 diabetes in SHR caused important
clinical decline of the animals that persisted until the end of the experiment.

3.1. Blood glucose

As expected, glycemia was strongly high in diabetic groups (five-fold increase). Since
the day after streptozotocin administration diabetic rats were persistently hyperglycemic
 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248 237

Fig. 2. Body mass evolution during the experiment (mean) (SHR = spontaneously hypertensive rats,
Db = streptozotocin diabetic animals). In signaled cases, when compared, p ≤ 0.05, if: (a) when compared with
SHR group, (b) with SHR-Db group, (c) with SHR-Db-olive oil group, (d) with SHR-Db-palm oil group.

(mean ± S.D., SHR-Db: 494.2 ± 128.7 mg/dl, SHR-Db-olive oil: 494.8 ± 64.0 mg/dl, SHR-
Db-palm oil: 349.6 ± 169.4 mg/dl, SHR-Db-fish oil: 486.2 ± 105.5 mg/dl) while pure SHR
were normal glycemic (96.4 ± 8.5 mg/dl). The edible oils administration did not change the
plasma glucose levels in all groups of rats.

3.2. Biometry

No difference among the groups was observed to both the naso-anal length and the LV
mass index.

3.2.1. Body mass

Diabetic rats suffered of polyuria, polydipsia, polyphagia and significant reduction in
body mass. Fig. 2 shows the body mass evolution during the experiment. Non-diabetic
SHR showed higher body mass than diabetic SHR. SHR-Db-palm oil group showed an
intermediary body mass between the other treated diabetic SHR and the SHR.

3.2.2. Blood pressure

Fig. 3 shows the BP evolution during the experiment that was significantly lower in
diabetic SHR than in non-diabetic SHR. As a rule, SHR showed higher BP levels in all
238 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248

Fig. 3. Systolic blood pressure (mean) (SHR = spontaneously hypertensive rats, Db = streptozotocin diabetic ani-
mals). In signaled cases, when compared, p ≤ 0.05, if: (a) when compared with SHR group, (b) with SHR-Db
group, (c) with SHR-Db-olive oil group, (d) with SHR-Db-palm oil group.

weeks than non-diabetic SHR. However, the edible oil treated groups showed intermediary
BP levels between untreated non-diabetic and diabetic SHR.

3.3. Myocardial structure

The myocardium in SHR showed the characteristic alteration due to overload (cardiomy-
ocyte hypertrophy) and ischemia (perimysial and interstitial fibrosis) (Fig. 4A). In diabetic
SHR, these lesions were also observed with frequent inflammatory regions (Fig. 4B) and
middle arteriolar hypertrophy with endothelial disruption (Fig. 4C). Diabetic SHR receiving
edible oils showed a less dramatic myocardial alteration than untreated diabetic SHR, even
if the animals taking olive oil showed inflammatory infiltrate in some regions (Fig. 4D), the
cardiomyocyte hypertrophy was less important and the cardiac interstitium and arterioles
showed practically normal structure in animals taking palm oil and fish oil (Fig. 4E and F,
respectively).
 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248 239

Fig. 4. Photomicrographs of the typical myocardial structure in the different groups (calibration bar = 50 ␮m). (A)
SHR group (stain picro Sirius red, bright field)—an extensive perimysial and interstitial fibrosis with inflamma-
tory cells (asterisk) surrounds hypertrophied cardiomyocytes. (B) SHR-Db groups (stain H-E)—a myocardium not
greatly injured shows inflammatory infiltrate (arrow). (C) SHR-Db group (stain Masson trichrome)—hypertrophied
cardiomyocytes and arteriole with perivascular fibrosis. The arteriole shows tunica media hypertrophy with hyper-
trophied smooth muscle cells and endothelial disruption. (D) SHR-Db-olive oil group (stain H-E)—longitudinal
view of moderately hypertrophied cardiomyocytes and an extensive inflammatory infiltrate (arrow). (E) SHR-Db-
palm oil group (stain H-E) and (F) SHR-Db-fish oil group (stain H-E)—both groups palm oil and fish oil show
myocardial structure almost normal, the cardiomyocytes are a little bigger in palm oil group than in fish oil group,
the microvasculature is abundant and no evident interstitial or perivascular fibrosis is observed.

3.4. Myocardial stereology

3.4.1. Connective tissue

The Vv[ct] measured the interstitial and perivascular fibrosis and was greater in SHR and
SHR-Db groups than in edible oils treated diabetic SHR. Compared to SHR or to SHR-Db
groups the sequence from major to minor in reducing Vv[ct] was fish oil = palm oil (less
50%, p < 0.008) > olive oil (less 35%, p < 0.008). The Vv[ct] was smaller in both SHR-Db-
240 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248

Fig. 5. Volume density of the myocardial connective tissue (bar graph, mean ± S.E.M.) (SHR = spontaneously
hypertensive rats, Db = streptozotocin diabetic animals). In signaled cases, when compared, p ≤ 0.05, if: [a] when
compared with SHR group, [b] with SHR-Db group, [c] with SHR-Db-olive oil group.

palm oil and SHR-Db-fish oil groups than SHR-Db-olive oil group (less 20%, p < 0.01) but
no difference occurred between SHR-Db-fish oil and SHR-Db-palm oil groups (Fig. 5).

3.4.2. Length density of the intramyocardial arteries

The hypertension or hypertension combined with diabetes impaired myocardial vas-
cularization. Compared to SHR or SHR-Db animals Lv[ima] was significantly greater in
diabetic SHR treated with edible oils (fish oil +180%, p < 0.008 > palm oil = olive oil +100%,
p < 0.008). The SHR-Db-fish oil group showed the highest Lv[ima] among the groups, more
than 35% higher than both SHR-Db-olive oil and SHR-Db-palm oil groups (p < 0.008).
There was not difference between SHR-Db-olive oil and SHR-Db-palm oil groups (Fig. 6).

3.4.3. Intramyocardial arteries/cardiomyocytes ratio

The ima/cmy ratio showed a similar tendency than Lv[ima] reinforcing those results
(Fig. 7).

3.4.4. Left ventricular cardiomyocyte mean cross-sectional area

SHR-Db-fish oil and SHR-Db-palm oil groups showed smaller A[cmy] than the other
groups except SHR-Db-olive oil group, around 20% smaller than the other groups.
 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248 241

Fig. 6. Length density of the intramyocardial arteries (bar graph, mean ± S.E.M.) (SHR = spontaneously hyperten-
sive rats, Db = streptozotocin diabetic animals). In signaled cases, when compared, p ≤ 0.05, if: [a] when compared
with SHR group, [b] with SHR-Db group, [c] with SHR-Db-olive oil group, [d] with SHR-Db-palm oil group.

There was not difference between SHR-Db-fish oil and SHR-Db-palm oil groups
(Fig. 8).

3.4.5. Number of left ventricular cardiomyocyte nuclei

This study showed that both non-diabetic and diabetic SHR lost cardiomyocytes during
the experiment. This fact was improved treating the diabetic SHR with edible oils. While
olive oil and palm oil showed a similar condition diminishing the loss of cardiomyocytes
(the N[cmyn] more than 50% higher than SHR-Db group) it was much greater in fish oil
treated animals (the N[cmyn] more than 130% higher than SHR-Db group). The N[cmyn]
was more than 40% higher in SHR-Db-fish oil group than in the other oil treated groups
and no difference was observed between SHR-Db-olive oil and SHR-Db-palm oil groups
(Fig. 9).

4. Discussion

The present results support the idea of beneficial effects of the long-term fish oil, palm
oil and olive oil supplementation (in the sequence from the more to the less beneficial oil
supplementation) for diabetic and hypertensive rats. The major effect of the oil intake was
242 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248

Fig. 7. Intramyocardial arteries to cardiomyocytes ratio (bar graph, mean ± S.E.M.) (SHR = spontaneously hyper-
tensive rats, Db = streptozotocin diabetic animals). In signaled cases, when compared, p ≤ 0.05, if: [a] when
compared with SHR group, [b] with SHR-Db group, [c] with SHR-Db-olive oil group, [d] with SHR-Db-palm oil
group.

observed reducing BP, enhancing myocardial microvasculature and, consequently, decreas-

ing the loss of cardiomyocytes, but no effect reducing blood glucose was observed.
Previous findings have already demonstrated anti-inflammatory [25] and hypotensive
effects caused by essential fatty acids [26], n-3 PUFA [27] and DHA [12], suggesting their
use in the prevention and treatment of hypertension [10] or diabetes [28].
The diabetes caused by streptozotocin administration increases fat mobilization in skele-
tal muscle [29] inducing significant weight loss [30] as was observed in the present study.
The treatment with edible oils, mainly palm oil, minimized the weight loss of diabetic ani-
mals. Palmitic acid, a dietary lipid derived from palm oil is saturated, but it is not a substrate
or a competitor of the hepatic delta-6 desaturase activity [31]. Palm oil is rich in antioxidant
vitamins and has beneficial effects in oxidative stress associated with hypertension and
arterial thrombosis [32]. However, by inducing their own oxidation, PUFAs are utilized for
fat storage within the muscle cell to a lesser extent than saturated fatty acids [33].
Streptozotocin-induced diabetes reduces BP independent of weight loss in SHR, and
could be the result of peripheral vasodilatation [34]. The BP-lowering effect of olive oil
supplementation [35], which was not confirmed by us in a previous investigation [10],
was only partial in this study. Fish oil administration was more efficient than palm and
olive oil in reducing BP of diabetic SHR, whereas palm oil showed similar BP levels
to untreated diabetic SHR. The fish oil enhances endothelium-dependent relaxation by
 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248 243

Fig. 8. Cardiomyocyte mean cross-sectional area (bar graph, mean ± S.E.M.) (SHR = spontaneously hypertensive
rats, Db = streptozotocin diabetic animals). In signaled cases, when compared, p ≤ 0.05, if: [a] when compared
with SHR group, [b] with SHR-Db group, [c] with SHR-Db-olive oil group.

replacement of endogenous arachidonic acid and suppression of the concomitant release of

vasoconstrictor prostaglandins from the endothelium [36]. Another possible mechanisms
for DHA BP-lowering effects is the reduction of vascular reactivity to norepinephrine,
blunting of the rennin–angiotensin–aldosterone system by decreasing adrenal synthesis of
aldosterone, changes in renal arachidonic metabolism, modulation of calcium release and
influx in vascular smooth muscle cells, and activation of vascular ATP-sensitive potassium
channels by vasodilatory prostanoids [12,37].
The myocardial interstitial alteration due to hypertension and diabetes was measured
by the volume density of connective tissue. The reparative fibrosis was typical in present
SHR animals [38,39] and was added of inflammatory infiltrate and arteriolar hypertro-
phy with perivascular fibrosis in diabetic SHR. There is good evidence that EPA and
DHA in fish oil abolished pro-inflammatory prostaglandin E2 production by inhibiting
the interleukin L1 beta-induced production of cyclooxygenase-2 mRNA [27]. Palm oil has
an unsaturated-to-saturated fatty acid ratio close to one and rich in antioxidant vitamins
and reduces oxidative stress and attenuates hypertension by mechanisms involving changes
in endothelium-derived factors [40]. EPA and DHA are known natural ligands of perox-
isome proliferator activated receptors (PPARs) and evidences suggest that PPAR ligands
may reduce the inflammatory response by attenuating the production of pro-inflammatory
244 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248

Fig. 9. Number of cardiomyocytes in the left ventricle (bar graph, mean ± S.E.M.) (SHR = spontaneously hyper-
tensive rats, Db = streptozotocin diabetic animals). In signaled cases, when compared, p ≤ 0.05, if: [a] when
compared with SHR group, [b] with SHR-Db group, [c] with SHR-Db-olive oil group, [d] with SHR-Db-palm oil
group.

cytokine [41] and n-3 PUFA may represent a class of naturally occurring, low-toxicity,
PPAR ligand with potent anti-inflammatory properties [42]. These arguments are in favor
of the present findings in streptozotocin diabetic (Db) genetic hypertensive rats; palm oil
and fish oil supplementation reduced BP and showed anti-inflammatory effects offsetting
adverse structural remodeling of cardiac tissue.
A major perspective in cardiac structure treating hypertension and diabetes is to improve
microvasculature. In the present study, diabetic SHR treated with fish oil supplementation
showed the best indicators of myocardial microvasculature among the groups of animals,
palm and olive oils groups were less beneficial than fish oil in this treatment and the length
density of intramyocardial arteries, as well as the ratio between these arteries and the car-
diomyocytes measured this efficiency. These findings suggest that any edible oil treatment
is better than no treatment. The long-chain polyenoics of EPA and DHA are reported to have
several beneficial actions on the vasculature, including incorporation of PUFA into vascular
membranes and resulting changes in membrane fluidity that may inhibit the expression of
 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248 245

adhesion molecules [43], proliferation of smooth muscle cells and change the activity of
several ion channels [44]. In addition, it has been proposed that long-chain PUFAs serve as
endogenous regulators of angiotensin converting enzyme activity, superoxide ion, endoge-
nous nitric oxide (NO) generation, and TGF-beta expression. Further, long-chain PUFAs
have actions similar to statins, inhibit (especially n-3 fatty acids) cyclooxygenase activity
and suppress the synthesis of pro-inflammatory cytokines, and activate the parasympathetic
nervous system, all actions that reduce the risk of major vascular events [45].
In the present study, fish oil treated diabetic SHR showed the smallest cardiomyocyte
size and the greatest cardiomyocyte number among the treated groups. Although some
cardiomyocytes can be binucleated during prenatal development [46] and binucleation of the
cardiomyocytes leads to inconsistencies in determining the cardiomyocyte number [47–49].
Mitotic activity of cultured cardiac muscle cells has shown that only 10–15% of cells fail
to complete cytokinesis, which become binucleated cells. The remaining 85–90% of the
mitotic cardiomyocytes complete cytokinesis and produce two daughter cells [50].
Usually the olive oil supplementation (except to cardiomyocyte size) and the palm oil
supplementation showed intermediary action between fish oil treatment and no treatment.
Linked normally with cardiomyocyte hypertrophy is relative ischemia due to the increased
artery-to-cardiomyocyte distance, which is a sufficient stimulus to cardiomyocyte apop-
tosis and necrosis [51,52]. Once again, we emphasize that NO plays an important role in
attenuating cardiac remodeling and apoptosis via suppression of oxidative stress-mediated
signaling pathways [53] and the endogenous NO generation is enhanced by long-chain
PUFAs [45].
Recently, a group identified a novel mediators generated from EPA and DHA that dis-
played potent bioactions were first identified in resolving inflammatory exudates [54,55]
and in tissues enriched with DHA [55,56], called resolvins and docosatriens, the activity of
which may in part explains the beneficial effects of n-3 fatty acids. Resolvins (resolution
phase interaction products) and docosatrienes demonstrate potent anti-inflammatory and
immunoregulatory actions. The compounds derived from eicosapentaenoic acid carrying
potent biological actions are designated E series, given their EPA precursor, and denoted
as Resolvins of the E series, and those biosynthesized from the precursor docosahexaenoic
acid are Resolvins of the D series. Bioactive members from DHA with conjugated triene
structures are docosatrienes that are immunoregulatory [55,56], and neuroprotective and are
termed neuroprotectins. Human Resolvin E1 binds to a G protein-coupled receptor called
ChemR23 that is expressed on leukocytes, and inhibits the migration of these cells to sites
of inflammation. In previous studies, aspirin promoted the cyclooxygenase-2-dependent
conversion of n-3 fatty acids to a precursor of Resolvin E1 [57–59]. The idea of anti-
inflammatory lipids effects on a new and more relevant therapeutic footing.

5. Conclusions

The long-term intake of edible oils had beneficial effects in reducing BP and offsetting
the usual cardiac adverse remodeling of streptozotocin-induced diabetic SHR. These effects
were marked with fish oil intake, and less marked with palm oil and olive oil, in this sequence.
The fish oil used in this study contained 31 mol% of EPA and 26 mol% of DHA. Present
246 F.J. Medeiros et al. / Prostaglandins & other Lipid Mediators 78 (2005) 231–248

findings are in favor of the supplementary edible oils administration, in addition to the
regular pharmacological treatment, to the situation of diabetes combined to hypertension.

Acknowledgements

The authors would like to thank Mrs. Tathiany de Souza Marinho and Mrs. Ana Claudia
Viana Soares for their technical assistance in material preparation. Dr. Rosemar Antoni-
assi kindly made the chemical analysis of the fish oil in EMBRAPA (Empresa Brasileira
de Pesquisas Agropecuarias), Rio de Janeiro, Brazil. This study was partially supported
by grants to the Laboratory of Morphometry and Cardiovascular Morphology from the
Brazilian National Council (CNPq) and Rio de Janeiro Foundation (FAPERJ).

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