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Unit of Physiology, University of Jaén, 23071 Jaén, Spain;
*
Department of Biochemistry and Molecular Biology, Medical School, University of Granada, Granada, Spain; and
Novartis Pharma, CH-4002, Basel, Switzerland
1To whom correspondence should be addressed. E-mail: msanchez{at}ujaen.es.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • aminopeptidases • dietary fat saturation • cholesterol • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. Hormonal changes in serum
may thus be reflected in this enzymatic activity. Significant changes
in serum AP activities during the estrous cycle of rats have been
reported (2)
. Orchiectomy and ovariectomy modified AP
activities significantly (3)
and cholesterol and steroids
affected AP activities in vitro (3
,4)
. These results have
suggested a role for these substances in the regulation of serum AP
activities.
A high intake of monounsaturated fat has been proposed to decrease the
incidence of cardiovascular disease and hypertension (5)
.
However, the mechanisms that underlie this effect are unknown. In
addition, increasing saturation of dietary fat resulted in increasing
plasma total cholesterol concentration (6)
and systolic
and diastolic blood pressures (7)
.
Aminopeptidases also play a major role in the regulation of the
renin-angiotensin system (1)
. In this system,
angiotensin (Ang) III is produced from Ang II by AP A or A-like
activity. Ang III is also produced from Ang I through the production of
des-Asp1-Ang I, which is further converted to Ang
III by the action of angiotensin-converting enzyme. Because GluAP
has been ruled out as the particulate enzyme that degrades Ang I to
des-Asp1-Ang I, another enzyme (AspAP) with
higher affinity for Asp-2-naphthylamide than Glu-2-naphthylamide must
be responsible (8)
. Ang III is further converted to Ang IV
by AP B (ArgAP) or AP M (AlaAP) (9)
.
We demonstrated previously that cholesterol selectively increased in
vitro the activity of GluAP and AspAP (aminopeptidase A activity), two
aminopeptidases directly related to Ang I and Ang II metabolism. In
contrast, there was no change in activity of other AP such as AlaAP and
ArgAP, which are involved in Ang III metabolism, CysAP, described as
vassopresin-degrading activity and pGluAP, described as
tirotropin-releasing hormone (TRH) or gonadotropin-releasing
hormone (GnRH)-degrading activity (3
, 4)
. In vitro
cholesterol selectivity prompted us to investigate the effect in vivo
of different degrees of dietary fatty acid saturation on serum
aminopeptidase activities, including angiotensinase activities and
others that are not related to angiotensin metabolism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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) containing 2.4 g/100 g of the respective experimental oil:
sunflower oil (SFO; n = 9), fish oil (FO; n
= 10), olive oil (OO; n = 8), lard (L;
n = 10) or coconut oil (CO; n = 9).
Mice were monitored for food intake and weight (data not shown). After
the feeding period, mice were killed under equithensin anesthesia (2
mL/kg body).
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. Supernatant (10 µL) was incubated for
30 min at 37°C with 1 mL of the substrate solution (21.4 mg/L
AlaNNap, 33.5 mg/L ArgNNap or 56.3 mg/L CysNNap), 0.1 g/L bovine serum
albumin (BSA) and 0.1 g/L dithiothreitol (DTT) in 50 mmol/L phosphate
buffer, pH 7.4, for AlaAP and ArgAP, and 50 mmol/L HCl-Tris buffer,
pH 6, for CysAP.
pGluAP was measured in a fluorogenic assay using pGluNNap as the
substrate, according to the modified method of Schwabe and McDonald
(11)
. Supernatant (10 µL) was incubated for
120 min at 37°C with 1 mL of substrate solution (25.4 mg/L pGluNNap,
0.1 g/L BSA, 0.1 g/L DTT and 378 mg/L EDTA in 50 mmol/L of phosphate
buffer, pH 7.4).
AspAP was determined fluorometrically with AspNNap as the substrate,
according to the method of Cheung and Cushman (12)
modified as follows: 10 µL of each supernatant was
incubated for 120 min at 37°C with 1 mL of the substrate solution
(25.8 mg/L, AspNNap, 0.1 g/L BSA, 0.1 g/L DTT and 394 mg/L
MnCl2 in 50 mmol/L HCl-Tris buffer, pH
7.4).
GluAP was also determined in a fluorometric assay using GluNNap as
the substrate according to the method of Tobe et al. (13)
modified as follows: 10 µL of each supernatant was
incubated for 120 min at 37°C with 1 mL of the substrate solution
(27.2 mg/L GluNNap, 0.1 g/L BSA, 0.1 g/L DTT and 5.55 g/L
CaCl2 in 50 mmol/L HCl-Tris, pH 7.4).
All of the reactions were stopped by adding 1 mL of 0.1 mol/L acetate
buffer (pH 4.2). The amount of ß-naphthylamine released as a result
of enzymatic activity was measured fluorometrically at 412 nm emission
wavelength with 345 nm excitation wavelength. Proteins were quantified
in triplicate by the method of Bradford (14)
with BSA as a
standard. Specific aminopeptidase activities were expressed as pmol of
AlaNNap, ArgNNap, CysNNap, AspNNap, pGluNNap or GluNNap hydrolyzed per
minute per milligram of protein. Fluorogenic assays were linear with
respect to time of hydrolysis and protein concentration. All chemical
products were supplied by Sigma Chemical. For statistical analysis, we
used one-way (ANOVA) with post-hoc comparisons using Tukey’s
test. Differences with P-values < 0.05 were considered
significant.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, and conjugated linoleic acid has
been found to lower body fat and weight in mice (16)
. The
contrast with the present results could be due to differences in age or
strain of mice studied. Although we used male Balb/C mice and started
with the defined diets at 1–2 wk, West et al. (16)
used
male inbred AKR/J mice and started the study at 5 wk.
Serum total cholesterol levels were 46% higher (P < 0.05) in mice fed diets containing saturated oils (L and CO) than in
those fed diets containing SFO (Fig. 1
). AspAP (P < 0.01) and GluAP (P < 0.01) were three- and fourfold greater, respectively, in mice fed CO
than in those fed SFO (Fig. 2
). The two serum aminopeptidase A activities increased progressively
with the degree of saturation of the fatty acid used in the diet. Mice
fed the diet containing CO differed significantly (P < 0.01) from those fed diets containing SFO and FO in AspAP and GluAP
activities (Fig. 2)
. The other activities measured in this study
(AlaAP, ArgAP, CysAP and pGluAP) did not differ among groups (data not
shown). Interestingly, the relatively low amounts of the various fats
(2.4 g/100 g) added to the diets in this study exerted notable effects
on cholesterol and aminopeptidase. This suggests that Balb/C mice, in
particular, may be an excellent model for these fat studies.
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,4)
, raising the possibility that
these substances create a biochemical environment that regulates the
activity of these enzymes. These earlier studies demonstrated that, in
vitro, cholesterol increased serum AspAP and GluAP activities.
Therefore, the present results may be due in part or indirectly to an
increase in serum total cholesterol, as a result of increasing
saturation of dietary fat. However, it should be take into account that
the origin of serum aminopeptidases under physiologic conditions is
unknown, and there is no evidence for active secretion from one or more
tissues. It has been proposed that circulating GluAP originates from
autolysis of the membrane-bound enzyme in several tissues,
including the kidney, where it is especially abundant
(17)
. This may be analogous to the origin in serum of
angiotensin-converting enzyme, which is originally
membrane-bound but becomes soluble in serum by autolysis
(18)
. Because the composition and physical properties of
membranes can be modified by dietary fat (19
,20)
,
modifications in autolysis may account for the changes observed in
membrane-bound AspAP and membrane-bound GluAP activities,
depending on the degree of dietary fat saturation.
In accordance with the present results, an increase in aminopeptidase A
activity suggests a heightened metabolism of Ang II, which leads to an
increase in Ang III formation. Therefore, if aminopeptidase A activity
is modified by the degree of dietary fat saturation, its substrates,
such as Ang I and Ang II, and its metabolic products, such as Ang III
and des-Asp1-Ang I, may also be modified.
Consequently, their actions in the control of blood pressure and other
physiologic functions may be similarly affected. It was demonstrated
recently that the fat saturation of the diet also influences other
enzymes such as esterases (21)
. Taken together, these
results suggest that dietary fat saturation has a wide range of effects
on various enzyme systems.
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FOOTNOTES |
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Manuscript received September 11, 2000. Revision accepted January 25, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Kudchodkar B. J., Lacko A. G., Dory L., Fungwe T. V. Dietary fat modulates serum paraoxonase 1 activity in rats. J. Nutr. 2000;130:2427-2433
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