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Relationship of angiotensinase and
vasopressinase enzymatic activities between
hypothalamus and plasma in aged rats by
high-fat diet

Germán Domínguez-Vías*, Ana Belén Segarra, Manuel
Ramírez-Sánchez, Sara Jiménez-Serrano

All Res. J. Biol., 2016, 7, 20-27

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ARTICLE

Issue 2, Vol 7, 2016, 20-27

Relationship of angiotensinase and vasopressinase enzymatic activities
between hypothalamus and plasma in aged rats by high-fat diet

Germán Domínguez-Vías1*, Ana Belén Segarra1, Manuel Ramírez-Sánchez1, Sara Jiménez-
Serrano1

1Unit of Physiology, University of Jaén, Jaén, Spain. *Correspondence to: Dr. Germán Domínguez-Vías,
Physiology Unit, University of Jaén, 23071 Jaén, Spain, e-mail: german.dominguez@uca.es

Graphical Abstract

Abstract: High-fat diets are associated with the development of hypertension. However, a high intake of
monounsaturated fat has been proposed to be a dietary factor that can decrease the incidence of hypertension.
The renin-angiotensin system (RAS) and vasopressin interact to regulate blood pressure at central and peripheral
level. In this study, we investigated the effect of different degrees of dietary fatty acid saturation in the control
of RAS and vasopressin on brain-blood. To improve our understanding of their interaction and their
relationship, we analyzed angiotensin- and vasopressin-metabolizing activities in hypothalamus and plasma,
collected from old Wistar rats fed for 24 weeks with diets enriched with extra virgin olive oil (monounsaturated
fat) or butter plus cholesterol (saturated fat) compared with a standard diet. We found no angiotensinase and
vasopressinase activities in hypothalamus and plasma, with no significant correlations between enzymatic
activities in either region. Therefore our results do not support the beneficial influence of extra virgin olive oil to
regulate blood pressure on the central or systemic levels. Furthermore, substrates downstream of vasopressin
and the RAS may be similarly unaffected, and given the previously described potential of the Mediterranean diet
as a tool in the treatment of hypertension, further studies need to be done in order to clarify the therapeutic
mechanism.

Keywords: angiotensinases, vasopressinase, renin-angiotensin system, brain-blood connection, dietary fat

All Res. J. Biol, 2016, 7, 20-27

Introduction

Dietary fat intake determines the fatty acid composition of
cell membranes and plays a well-recognized role in
cardiovascular risk and the development of cardiometabolic
disease1. Moreover, diet composition may also influence
brain activity as the dietary fat modifies the brain membrane
fluidity2,3. A high intake of monounsaturated fat has been
proposed to decrease the incidence of hypertension4-6. In
addition, increasing saturation of dietary fat resulted in
increasing plasma total cholesterol concentration7 and
systolic and diastolic blood pressures8. The systemic and the
local renin-angiotensin system (RAS), and vasopressin in
brain9, interact together to regulate blood pressure (BP) by
various endocrine and autonomic mechanisms10. The
hypothalamic stimulatory effect of angiotensin (Ang) III
(Ang III) on vasopressin release has been clearly
demonstrated11. The hypothalamus is known to integrate
behavioral, endocrine, neuroendocrine, and autonomic
responses (including those that regulate cardiovascular
function) to maintain homeostasis12. We hypothesized that
the direct and/or indirect relationship between the
hypothalamus and plasma, both of which regulate BP, may
be mediated to some extent by angiotensinase and
vasopressinase enzymatic activities and the metabolism of
their corresponding peptidergic substrates.

In order to analyze the link between RAS and vasopressin in
the control of BP, as well as the relationship between the
hypothalamus and plasma, we have studied the metabolism
of (A) Ang I to Ang 2-10, (B) Ang II to Ang III, (C) Ang III
to Ang IV and (D) the metabolism of vasopressin by
measuring the activities of (A) aspartyl aminopeptidase
(AspAP), (B) glutamyl aminopeptidase (GluAP), (C) alanyl
aminopeptidase (AlaAP), arginyl aminopeptidase (ArgAP),
and (D) cystinyl aminopeptidase (CysAP), respectively13,14.
Additionally, we observed the activities of angiotensinase
and vasopressinase in plasma and their soluble and
membrane-bound fractions from the anterior hypothalamus.
In our model, aged male Wistar rats were fed with high-fat
diets (HFD) supplemented with different degrees of dietary
fatty acid saturation, such as olive oil rich in
monounsaturated fatty acids (MUFAs), or butter plus
cholesterol rich in saturated fatty acids (SAFAs). Enzymatic
activities were determined by fluorimetry using arylamide
derivatives as substrates, to determine the effect of dietary fat
on RAS, vasopressin, and ultimately blood pressure
regulation.

Experimental Methods

Animals and treatments

17-week-old male Wistar rats were purchased from Harlan
Ibérica (Barcelona, Spain). The animals were allowed free
access to food and water during 24 weeks and were
maintained on a 12 hours light/dark cycle in a controlled
temperature (20-25ºC) and humidity (50 ± 5%) environment.

Mean body weight was ~495 g at beginning of the study.
Experimental procedures for animal use and care were in
accordance with European Communities Council Directive
2010/63/UE and Spanish regulation RD 53/2013. Rats were
randomly assigned into three groups (5-6 animals per group)
as follows. In standard diet (S) group, rats were fed with a
commercial diet for experimental control. In HFD groups,
one group of mice (VOO) was fed with a diet supplemented
with 20% monounsaturated fat (virgin olive oil, VOO), and
the last group (Bch) was fed a diet supplemented with 20%
saturated fat (butter) plus 0.1% cholesterol . The HFD groups
were isocaloric. Food composition in different groups and
nutritive value are shown in supplemental tables (Appendix
1 and 2). Twenty four weeks after the feeding period, a
sample of blood was collected for peptidase activities assay,
then the animals were sacrificed under equithensin (2ml/kg
body weight) and perfused with saline solution through the
left cardiac ventricle. The blood was centrifuged for 10 min
at 2,000 g to obtain the plasma, which was stored at -20ºC.
Hypothalamus tissue was also collected and dissected as
previously described10,15. Briefly, the brain was quickly
removed (less than 60 s) and cooled in dry ice. The
hypothalamus (pooled left and right) was dissected according
to the stereotaxic Paxinos and Watson atlas16. The selected
area was between 7.7 mm and 3.7 mm anterior to the
interaural line.

Sample preparation for enzyme activity assay

Samples from plasma were directly used for the peptidase
activity assay. Samples from the hypothalamus were quickly
removed and frozen in dry ice. To obtain the soluble fraction,
tissue samples were homogenized in ten volumes of
hypoosmolar medium (10 mM HCl-Tris buffer, pH 7.4) and
ultracentrifugated (100,000 g for 30 min at 4ºC). The
resulting supernatants were used to measure soluble (sol)
angiotensinase and vasopressinase activities and protein
content, assayed in triplicate. To solubilize membrane
proteins, pellets were rehomogenized in HCl-Tris buffer (pH
7.4) plus Triton X-100 (1%). After centrifugation (100,000 g
for 30 min at 4ºC), supernatants were used to measure
membrane-bound (mb) activity and proteins, also in
triplicate. To ensure the complete recovery of activity,
detergent was removed from the medium by adding to the
samples adsorbent polymeric Biobeads SM-2 (100 mg/ml)
purchased from Bio-Rad (Richmond, VA, USA) and shaking
for 2 h at 4ºC17.

Peptidase activities assay

Sol and mb angiotensinase and vasopressinase activities were
determined fluorometrically using the arylamide derivatives
aspartyl-, glutamyl-, alanyl-, arginyl-, and cystinyl-β-
naphthylamide (-β-NNap) as substrates according to the
method of Ramírez18. Briefly, for AlaAP and ArgAP assay,
10 µL of each supernatant were incubated for 30 min at 37ºC
with 100 µL of substrate solution (100 µM Alanyl- or
Arginyl-β-NNap), 1.5 mM albumin seric bovine (BSA), and
0.65 mM dithiothreitol (DTT) in 50 mM phosphate buffer pH

All Res. J. Biol, 2016, 7, 20-27

7.4). For AspAP assay, 10 µL of each supernatant were
incubated for 30 min at 37ºC with 100 µL of substrate
solution (100 µM Aspartyl-β-NNap), 1.5 mM albumin seric
bovine (BSA), and 3·10-8 mM CaCl2 in 50 mM Tris-HCl
buffer pH 7.4). For GluAP assay, 10 µL of each supernatant
were incubated for 30 min at 37ºC with 100 µL of substrate
solution (100 µM α-Glutamyl-β-NNap), 1.5 mM albumin
seric bovine (BSA), 0.65 mM DTT, and 50 mM MnCl2 in 50
mM Tris-HCl buffer pH 7.4). Finally, for CysAP assay, 10
µL of each supernatant were incubated for 30 min at 37ºC
with 100 µL of substrate solution (100 µM Cystinyl-β-
NNap), 1.5 mM albumin seric bovine (BSA) and 0.65 mM
DTT in 50 mM Tris-HCl buffer pH 6.0). All the reactions
were stopped by adding 100 µl of 0.1 M acetate buffer (pH
4.2). The amount of β-NNap released as a result of enzymatic
activity was measured fluorometrically at 412 nm emission
wavelength with 345 nm excitation wavelength. Activities of
specific peptidases were expressed as pmol of β-NNap
hydrolyzed per minute and per milligram of protein.
Fluorogenic assays were linear with respect to time of
hydrolysis and protein concentration. Protein concentration
was determined colorimetrically according to Bradford
method19 with BSA as standard. All chemical products were
supplied by Sigma (St. Louis, MO USA).

Statistical analysis

Statistical analysis was performed by one-way ANOVA
followed by Tukey post-hoc test for multiple comparisons.
Relations between variables were determined by Pearson’s
coefficients of correlation analysis. Statistical significance
was evaluated with Sigmaplot v.11 software (Systat
Software, Inc., San Jose, CA, USA). A P-value below 0.05
was considered to be statistically significant. All results were
expressed as mean ± standard error.

Results

Results are represented in Figure 1-6. In spite of the
importance of MUFAs to maintain normal total body weight
(Figure 1A), plasma triglycerides (data not shown) and
cholesterol levels (Figure 1B) and, fundamentally, BP
(Figure 2), there were no significant differences in systemic
angiotensinase (AlaAP, ArgAP, AspAP, GluAP) and
vasopressinase (CysAP) activities in plasma (Figure 3 and
4). Likewise, there were no significant differences in either
soluble nor membrane bound angiotensinase (Figure 5) and
vasopressinase (Figure 6) activities in hypothalamus.

Therefore, the type of fat added to the diet –
monounsaturated or saturated – did not seem relevant in the
metabolic control of central and systemic RAS and
vasopressin. Besides, significant correlations were not
observed between fractions of angiotensinase and
vasopressinase activities from hypothalamus with their
homologs in plasma.

Additionally, the hydrolysis of amino acid naphthylamides
by aminopeptidases from hypothalamus and plasma also did
not show significant correlation with plasma cholesterol
levels.

Figure 1. A. Total body weight after feeding period. B. Blood
cholesterol ratio between total cholesterol and high density
lipoprotein-cholesterol plasma fraction (HDL-C) after feeding
period. A higher ratio means a higher risk of heart disease. ** (p
<0.001) versus standard diet (S), # (p < 0.05) versus virgin olive oil
diet (VOO).

Figure 2. Systolic blood pressure (SBP) after feeding period. * (p
<0.05) versus standard diet (S), # (p < 0.05) versus virgin olive oil
diet (VOO).

All Res. J. Biol, 2016, 7, 20-27

Figure 3. Systemic angiotensinase activities. No significant
differences were found in plasma (A) AlaAP, (B) ArgAP, (C)
AspAP, and (D) GluAP between dietary groups after the feeding
period.

Figure 4. Systemic vasopressinase activity. No significant
differences were found in plasma CysAP between dietary groups
after the feeding period.

Discussion

In this study we analyzed the potential role dietary fatty acid
saturation in the control of RAS and vasopressin on brain-
blood connection. After 24 weeks of treatment with diets
supplemented with monounsaturated fat (olive oil, rich in
MUFAs) or saturated fat (butter plus cholesterol, rich in
SAFAs), our results did not show diet-specific differences in
angiotensinase and vasopressinase activities in either plasma
or the hypothalamus. The two evaluated diets did not
significantly change aminopeptidase (AP) activity as
compared to the standard diet, and likewise there were no
significant correlations between metabolic activities in the
brain and blood, suggesting that the fatty acid composition of
the diet might not have influence on the function of the
hypothalamus-plasma connection.

Previous studies have also determined no changes in global
AP/neuropeptidase (e.g.: AspAP and GluAP) activity in
frontal cortex3,20 in adult male rats whose diets were
supplemented with fatty acids with varying degrees of
saturation, such as fish oil (rich in polyunsaturated fatty
acids, PUFAs), olive oil (rich on monounsaturated fatty
acids, MUFAs), and coconut oil (rich in saturated fatty acids,
SAFAs). The authors proposed that the types of lipids in the
diet affect the fluidity of the membrane, with increase or loss
of membrane-associated enzymes20. They observed that the
diet composition affects fatty acid distribution in the brain.
The change in fluidity may also affect the tertiary structure of
the enzyme embedded in the membrane. This may affect the
binding of the enzyme with its substrate2,20, which may
explain the positive or negative correlation between fatty acid
content and membrane-bound and soluble fraction of the
angiontensin- and vasopressin-degrading enzymes observed
in the previous work; however it was not possible to clarify it
in this current work.

All Res. J. Biol, 2016, 7, 20-27

Figure 5. Soluble (sol) and membrane-bound (mb) fractions of
central angiotensinase activities. No significant differences were
found in hypothalamus (A) AlaAP, (B) ArgAP, (C) AspAP, and (D)
GluAP between dietary groups after thefeeding period.

Figure 6. Soluble (sol) and membrane-bound (mb) fractions of
central vasopressinase activity. No significant differences were
found in hypothalamus CysAP between dietary groups after feeding
period.

Many studies reveal that a HFD can modify BP by
dysregulation of RAS and their regulatory enzymes: the
effect of the diet on peripheral enzymatic activity was clearly
demonstrated21,22. Our results showed a decrease of BP that
correlated with MUFAs, although that physiologic response
was not linked to enzyme activities in hypothalamus and
plasma. Other works23,24 described significant changes in
their angiotensinase and vasopressinase activities in central
and visceral tissues such as pituitary gland25, heart25, aorta25,
adrenal gland25, kidney25, liver26, and testis27,due to fat
saturation in the diet, but seemingly did not investigate
enzyme activity in the hypothalamus and plasma. The Bch
diet was the only treatment which determined increase in
body weight28, high serum triglyceride and cholesterol
levels28, systolic blood pressure28, serum nitrates and
nitrites28, and hepatic inducible nitric oxide (NO) synthase
(iNOS) expression28, however, these metabolic changes
produced by a diet supplemented with saturated fat did not
show an expected hypothalamus-plasma imbalance on the
RAS-vasopressin nexus. No significant correlations were
observed between the AP values and lipid profile;
nevertheless, previous results have suggested that cholesterol
influences serum AP activities29,30, including the enzymes of
study, raising the possibility that these compounds create a
biochemical environment that regulates the activity of these
enzymes. Therefore, the present results may be partially or
indirectly due to an increase in plasma total cholesterol, as a
result of increasing saturation of dietary fat.

Other authors15,31-33 showed that an increase in AspAP and
GluAP suggest a heightened metabolism of Ang II, which
leads to an increase in Ang III formation. Therefore, if both
angiotensinases are modified according to the degree of
saturated fat in the diet, their substrates, such as Ang I and
Ang II, and their metabolic products, such as Ang III and
des-Asp-Ang I, may also be modified. Consequently, their
roles in the control of BP and other physiologic functions
may be similarly affected. It has been demonstrated that the
fat saturation of the diet also influences other enzymes, such
as dipeptidyl peptidase-IV (DPP-IV)34,35 and gamma-

All Res. J. Biol, 2016, 7, 20-27

glutamyl transpeptidase (GGT)25,36,37. Taken together, these
results suggest that dietary fat saturation has a wide range of
effects on various enzyme systems, despite not having been
demonstrated in the hypothalamus and plasma.

It is well established that the RAS over-activity is connected
to the hypertension produced by saturated fat. Hypertension
models have demonstrated a concomitant reduction of
AspAP and therefore reduced formation of Ang 2-10, which
has been suggested to counteract Ang II31. Thus, the
reduction of Ang 2-10, together with a higher release of
vasopressin due to the increased availability of Ang III, may
contribute to the higher BP in L-NAME treated
spontaneously hypertensive rats (SHR)38. Despite this, it is
known that VOO treatment of SHR delays the decrease of
systolic BP, and also presents with decreased levels of NO
and 8-isiprostatones assayed in urine21. Coupled with these
results, a highly significant down-regulation in AspAP and
GluAP stimulates a higher formation of angiotensin 2-10 in
the renal cortex21, as well as, a higher availability of Ang II
in the renal medulla of animals fed a VOO diet than in
animals fed a standard diet21.

Our results showed that rats in the Bch group, but not the
VOO group, had increased BP28; however, the lack of
changes in hypothalamus and plasma activities in rats treated
with HFD suggested no involvement of
hypothalamus/plasma Ang III and Ang IV and determined
unaltered vasopressin. On the other hand, previous work
observed hypercholesterolemia and increased body weight, as
well as significantly increased serum ArgAP22 and decreased
GluAP24, in rats fed with a diet supplemented with VOO as
compared to the standard diet. Likewise, sol AlaAP and
ArgAP activities were increased in the brain31. These
findings show that a diet supplemented with olive oil
modifies certain AP activities in brain and serum, and these
results may reflect functional modifications in susceptible
endogenous substrates.

Oxytocin, together with vasopressin, is the principal substrate
of CysAP. The Zorad group showed that obesity is associated
with reduced plasma oxytocin due to increased peptide
degradation by liver and adipose tissue rather than changes in
hormone synthesis39. The use of polyunsaturated fatty acid,
such as fish oil, demonstrates higher levels of CysAP activity
in mice than in those that were fed diets containing saturated
oils (lard or coconut)40. Our previous results also highlighted
an increase mb CysAP in the liver with VOO26, but no
evidence of changes in vasopressinase activity was found in
the hypothalamus and plasma. Oxytocinase/vasopressinase
inhibition has been suggested as a candidate approach in the
therapy of obesity39,41-43.

Conclusion

All enzymatic activities found in hypothalamus and plasma
showed no significant differences in our three experimental
diet groups, which might indicate that if membrane changes
occur, they could not significantly be involved in
angiotensin-/vasopressin-degrading activities. Therefore, we
propose that the absence of correlations between these

enzymes and the type of fatty acids indicates that they might
not be involved in the modulation of RAS and cognitive
functions and, consequently, in the onset of hypertension and
possible neurodegenerative disorders as causes of this
disorder. Finally, our results do not support the beneficial
influence of virgin olive oil on central and systemic
cardiovascular function, despite previous evidence that the
Mediterranean diet plays a major beneficial role in lowering
BP. The present observation should be taken into account in
strategies for the prevention of such diseases and as future
diagnostic tools.

Acknowledgments

We thank to Rocío Díaz-Ríos (Writer’s First Aid) for English
language revision of this manuscript. This work was
supported by University of Jaén through “Plan de Apoyo a la
Investigación 2006-2008”.

Appendix (Supplemental Tables)

Appendix 1: Food composition in three different diets.

CHOW COMPOSITION

Ingredients (g/Kg) S VOO Bch
Casein
Methionine

-
-

162
3

Sucrose
Starch
Maltodextrin

-
-
-

288
160
100

Virgin olive oil
Butter
Cholesterol

-
-
-

200

234
10

Mineral-Vitamin
Correcting
Choline chloride

4
Fiber (Cellulose) - 40 40

TOTAL - 1000 1000

LIPID PROFILE (%)
Saturate
Monounsaturated
Polyunsaturated

25
21
54

13.5
73.7
8.4

50.5
23.4

Appendix 2: Nutritive value of different diets.

NUTRITIOUS VALUE OF THE DIET (%)
Diet type S VOO Bch
g Kcal g Kcal g Kcal

Proteina 16.5 20 16.5 14 16.5 14

Carbohydrates 60 72 55 48 55 48

Fats 3 8 20 38 20 38

Total energy
(Kcal/g) 3.410 4.740 4.740

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