The effects of L-arginine and L-NAME supplementation on redox-regulation and thermogenesis in interscapular brown adipose tissue
1
2
1
1,*
1 Department of Physiology, Institute for Biological Research,
`Sinia Stankovi
', University of Belgrade, Bulevar Despota
Stefana 142, 11060 Belgrade, Serbia and Montenegro
2 Institute of Zoology, Faculty of Biology, University of Belgrade,
Studentski trg 16, 11000 Belgrade, Serbia and Montenegro
* Author for correspondence (e-mail: koracb{at}ibiss.bg.ac.yu)
Accepted 22 September 2005
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Summary |
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Cold generally diminished both iNOS immunopositivity and protein level in IBAT, as well as the rate of apoptosis. Among groups acclimated to cold, higher iNOS immunopositivity and protein levels were detected only in the L-Arg-treated group. Furthermore, chronic L-Arg treatment increased IBAT mass and UCP1 protein content, while L-NAME had an opposite effect, decreasing both IBAT mass and UCP1 protein level, as compared to the control maintained at 4±1°C.
These data suggest that nitric oxide (NO) produced by iNOS could also contribute to overall NO-associated regulation of thermogenesis in IBAT. Namely, that iNOS, i.e. NO, in correlation with enhanced thermogenesis, additionally induced IBAT hyperplasia and UCP1 level compared to that induced by low temperature. Cooperative action of decreased apoptosis accompanied by increased tissue hyperplasia and UCP1 level, observed in IBAT of cold-acclimated rats, would be a way of meeting the metabolic requirements for increased thermogenesis.
Key words: interscapular brown adipose tissue, inducible nitric oxide synthase, nitric oxide, uncoupling protein 1, cold, apoptosis, MnSOD, glutathione, rat
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Introduction |
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Nitric oxide (NO) is a gaseous messenger molecule implicated in numerous
biological functions in both physiological and pathological conditions.
Production of NO is catalyzed by three NO synthase (NOS; EC 1.14.13.39)
isoforms (Förstermann et al.,
1995). The endothelial and neuronal NOS (eNOS and nNOS,
respectively) are regulated by second messengers and the third one, inducible
NOS (iNOS) is inducible in a wide range of cells and tissues. It has been
shown that brown adipocytes express the eNOS
(Giordano et all., 2002
) and
iNOS (Nisoli et al., 1997
) and
that NO produced by these isoforms is involved in regulation of BAT function
(Kikuchi-Utsumi et al., 2002
;
Nisoli et al., 1997
,
2003
). Recent findings
justified the role of NO in BAT non-shivering thermogenesis
(Saha and Kuroshima, 2000
). It
can mediate increased blood flow to BAT following noradrenergic stimulation
(Nagashima et al., 1994
;
Kikuchi-Utsumi et al., 2002
)
and take part in differentiation and proliferation
(Nisoli et al., 1998
) in brown
adipocyte cultures. An obligatory role of NO produced by eNOS in mitochondrial
biogenesis has been established very recently (Nisoli et al.,
2003
,
2004
;
Clementi and Nisoli, 2005
).
However, the ability of iNOS, i.e. NO, to enhance interscapular BAT (IBAT)
hyperplasia and UCP1 expression in IBAT of rats during cold acclimation has
not so far been examined. This prompted us to examine iNOS level in relation
to the IBAT hyperplasia and UCP1 protein level in IBAT of cold-acclimated rats
receiving L-Arg or l-NAME as a drinking liquid for 45 days.
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Materials and methods |
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The animals were killed by decapitation, the IBAT dissected out within 3
min after death, minced and thoroughly rinsed with physiological saline to
remove traces of blood. The tissue was homogenized at 0-4°C (using a
Ka/Werke Ultra/Turrax homogenizer, Janke and Kunkel, Stenssen, Germany) in
0.25 mol l-1 sucrose, 0.1 mmol l-1 EDTA and 50 mmol
l-1 Tris HCl buffer, pH 7.4. The homogenates were sonicated as
suggested by Takada et al.
(1982).
Activity of manganese superoxide dismutase (MnSOD; EC 1.15.1.1)
Superoxide dismutase (SOD) activity was examined by a modified method of
Misra and Fridovich (1972).
Total specific SOD and CuZnSOD activities after inhibition with 4 mmol
l-1 KCN were measured and MnSOD activity calculated. Enzymatic
activity was expressed in U mg-1 protein. SOD units were defined as
the amount of the enzyme inhibiting epinephrine oxidation by 50% under the
appropriate reaction conditions.
Determination of glutathione (GSH)
The content of glutathione (GSH) was examined in the tissue after
deproteinization with sulfosalicylic acid. Total GSH was measured by
enzyme-recycling assay after Griffith
(1980) and expressed in nmol
GSH g-1 tissue.
Immunohistochemistry
Immediately after dissection and washing, the IBAT samples were fixed in
10% formaldehyde at 4°C overnight and processed routinely for embedding in
paraffin. 5 µm thick serial IBAT sections were deparaffinized and
rehydrated. Immunoreactivity was assessed by the avidin-biotin-peroxidase
method (following the manufacturer's instructions; Santa Cruz Biotechnology,
Santa Cruz, CA, USA). The sections were incubated with 0.3%
H2O2 in methanol for 10 min at ambient temperature to
block endogenous peroxidase, followed by three 5 min washes in 0.015 mol
l-1 phosphate-buffered saline (PBS; pH 7.4) and incubated with 1.5%
normal goat serum (ABC Staining System, Santa Cruz Biotechnology) in PBS for
60 min atambient temperature to block non-specific sites. The primary antibody
against iNOS (Santa Cruz Biotechnology) was a polyclonal antibody produced in
rabbits. The sections were incubated with the primary antibody in PBS (diluted
1:200 v/v) overnight at 4°C, followed by two 5 min PBS washes, incubated
with 1:200 IgG biotinylated serum goat anti-rabbit (ABC Staining System, Santa
Cruz Biotechnology) in PBS for 60 min at ambient temperature, followed by two
5 min PBS washes. After that, AB reagent (ABC Staining System, Santa Cruz
Biotechnology) was added for 30 min at ambient temperature, followed by three
5 min PBS washes and incubation with 0.02% H2O2 and
0.075% diaminobenzidine (Sigma) in 0.05 mol l-1 Tris buffer, pH
7.6, for 10 min in a dark room. Rinsing in distilled water and counterstaining
with Hematoxylin completed the experimental schedule. Negative controls were
prepared by omitting the primary antibody.
SDS-PAGE and western blotting
For UCP1 and iNOS analyses by western blotting, proteins were dissolved
according to Laemmli (1970)
and 10 µg protein samples boiled and electrophoresed in 15% and 7.5%
SDS-polyacrylamide gel for UCP1 and iNOS analysis, respectively. After that
proteins were transferred onto nitrocellulose membranes. After a brief rinse
in TBS (200 mmol l-1 Tris, 1.5 mol l-1 NaCl, pH 7.4),
the membranes were incubated in blocking serum (TBS containing 5% BSA) for 1 h
at ambient temperature to block the unbound sites. The membranes were further
incubated with rabbit polyclonal antibody against UCP1 (Sigma-Aldrich Inc., St
Louis, MO, USA) and with rabbit polyclonal antibody against iNOS (Chemicon
International Inc., Temecula, CA, USA). The incubation with primary antibodies
was performed in TBS-T (0.2% Triton X-100 in TBS) with 5% BSA at 1:1000 v/v
with antibody against UCP1 and primary antibody against iNOS, as recommended
by the manufacturer (i.e. 5 µl ml-1), overnight in a cold room.
After multiple washes in TBS-T the membranes were incubated with horseradish
peroxidase-conjugated IgG secondary antibody (Santa Cruz Biotechnology) at
1:2000 v/v. For UCP1 detection, peroxidase activity was revealed using
4-chloro-1-naphthol and H2O2 as a substrate. For iNOS
detection, the membrane was covered by luminol-based chemiluminescent
substrate for 3 min. Immediately after, a piece of X-ray Hyperfilm MP
(Amersham API, Indianapolis, IN, USA) was placed over the blot and exposed for
1 min. The film than was developed, scanned and quantitative analysis of
immunoreactive bands was done by an ImageQaunt software (Piscataway, NJ, USA).
Volume is the sum of all the pixel intensities within a band; 1 pixel=0.007744
mm2.
Detection of apoptosis
IBAT sections 5 µm thick were used for immunohistochemical detection of
apoptosis by TUNEL labeling of the nuclei showing specific oligonucleotide
sequences resulting from DNA strand breaks. Staining was performed with the In
Situ Cell Death Detection Kit POD (Boehringer Mannheim, Germany) according to
the manufacturer's instructions.
Protein content was estimated by the method of Lowry et al.
(1951).
Student's t-test was used for data comparison between different
groups according to Hoel
(1966). The P<0.05
level was chosen as the point of minimal acceptable statistical
significance.
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Results |
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The iNOS immunoblot (Fig. 2A) confirmed the immunohistochemical results, showing very faint bands in all groups acclimated to cold but strong bands in the groups kept at ambient temperature. Nevertheless, among cold-acclimated groups, the strongest band was detected in the L-Arg-treated group. Also, the bands in both L-Arg-treated and L-NAME-treated groups maintained at ambient temperature were stronger than in the corresponding control. Quantification of iNOS bands by ImageQuant software (Fig. 2B) revealed that the iNOS protein level of cold-acclimated rats was significantly higher in L-Arg-treated groups than in the control (P<0.005), while the level of this protein in L-NAME-treated rats was decreased, but the difference was not statistically significant (Fig. 2B). In IBAT of both L-Arg- and L-NAME-treated rats maintained at room temperature, iNOS level was significantly higher than in the corresponding controls (P<0.025 and P<0.005, respectively).
The results of IBAT mass and protein concentration in different groups of animals are summarized in Table 1. It can be seen that IBAT mass, as well as protein content, were increased (P<0.005) in all groups exposed to low temperature in comparison with the corresponding controls kept at room temperature. The IBAT mass showed a rising trend in L-Arg-treated group acclimated to low temperature, whereas that of L-NAME-treated group acclimated to low temperature was significantly decreased (P<0.005) in comparison with the controls acclimated to low temperature.
|
Western blot analyses revealed that UCP1 occurred only in IBAT of rats acclimated to low temperature (Fig. 3A). Quantification of UCP1 bands by ImageQuant software showed a significantly higher UCP1 level in L-Arg-treated rats than in the control (P<0.005), while the level of this protein was significantly decreased in L-NAME-treated rats compared to the control (P<0.005; Fig. 3B). No UCP1 was detected in IBAT of animals of any group maintained at room temperature.
|
The changes in GSH content in IBAT are depicted in Fig. 5. As seen, GSH content in IBAT was significantly higher in all animals acclimated to low temperature in comparison with the corresponding controls kept at room temperature with different statistical significance. Among groups acclimated to cold, GSH content was significantly decreased in L-Arg-treated rats (P<0.025), but significantly increased in L-NAME-treated cold-exposed animals in comparison with the control acclimated to low temperature (P<0.025).
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Discussion |
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Our results show that iNOS isoform is expressed in IBAT and that long-term exposure to cold led to a decrease in the level of iNOS protein. Chronic treatment with L-Arg of animals exposed to cold resulted in an increased iNOS level and immunopositivity compared to the control acclimated to cold, while L-NAME had an opposite effect, i.e. it caused decreased iNOS levels and immunopositivity as compared to cold-acclimated control.
We also show that the IBAT mass was significantly increased in animals
acclimated to cold. This is in accordance with the data of the others
(Bukowiecki et al., 1986;
Puerta et al., 1990
), as well
as with our earlier results
(Buzad
i
et al.,
1999
), demonstrating that IBAT of cold-acclimated rats undergoes
hyperplastic changes. The IBAT mass was increasing in L-Arg-treated
groups acclimated to low temperature compared to the corresponding controls,
and significantly decreased in L-NAME-treated groups acclimated to
cold in comparison with the cold-acclimated controls. This agrees well with
the results of Saha et al.
(1996
), who found that
L-NAME administered in drinking water for 4-6 weeks caused
decreased IBAT mass. In accordance with previous observations, we presumed
that NO additionally intensified the cold-induced increase of IBAT mass. This
is in accordance with Nisoli et al.
(1998
) and Kikuchi-Utsumi
(2002
), who reported that NO
(exogenous or generated by constitutive eNOS isoforms) plays a significant
role in brown adipocytes.
As an integral part of IBAT hyperplasia and in correlation with an enhanced
thermogenesis, the UCP1 level was increased upon exposure to cold.
Accordingly, we observed that UCP1 was expressed only in IBAT of rats
acclimated to low temperature, in agreement with the results of Ricquier et
al. (1983). Moreover, we
showed that UCP1 level in L-Arg-treated rats acclimated to cold was
significantly higher than in the corresponding control. In contrast, the UCP1
level was significantly decreased in L-NAME-treated animals
acclimated to low temperature compared to the control kept at the same
temperature. These results suggest that NO additionally induced UCP1, compared
to induction of UCP1 by cold. To our knowledge, this is the first evidence for
in vivo regulation of UCP1 expression by NO. Nisoli et al.
(1998
) observed that NO
increased UCP1 expression of cultured brown adipocytes. It is known that NO is
capable of interacting with many cellular targets including oxygen, superoxide
anion radical (O2.-), thiols, and particularly with
reduced glutathione (GSH; Beckman and
Koppenol, 1996
). We assumed that the effect of NO on UCP1
expression could be mediated by O2.- or GSH, but it does
not seem to be due to its interaction with O2.-. In
fact, it is known that uncoupling induced by thermogenesis acts to decrease
O2.- production
(Boveris et al., 1972
;
Cino and Del Maestro, 1989
;
Skulachev, 1994
). The
specificity of SODs, essential O2.- scavengers
(Fridovich, 1978
), for the
reaction with O2.- has frequently been used to probe for
the involvement of this radical in biological systems. Our previous studies
(Petrovi
et al., 1989
)
along with the findings of the others
(Koch et al., 1994
;
Perera et al., 1995
) showed
that MnSOD is easily induced by O2.- and different
agents, and more inducible than CuZnSOD. Accordingly, to check the hypothesis
that the effect of NO on UCP1 level was mediated by
O2.-, we determined MnSOD activity. MnSOD activity was
significantly decreased in rats acclimated to cold, possibly representing an
adaptive response due to long-term reduced production of
O2.- in IBAT mitochondria, as a consequence of UCP1
induction and uncoupling respiration from phosphorylation, which is the main
prerequisite of IBAT thermogenesis. Taken together, these results suggest that
in the regulation of UCP1 expression by NO, reaction with
O2.- is not the main pathway. Decreased level of GSH in
L-Arg-treated rats maintained at low temperature compared to the
control acclimated to the same temperature, suggests that another interaction
of GSH with NO took place.
Reduced glutathione (GSH) represents the most important non-enzymatic
intracellular antioxidant, the major function of which is to maintain cellular
homeostasis. Several authors have suggested that GSH is the prime
thiol-containing target for NO in a cell
(Kröncke et al., 1998;
Reichenbach et al., 2001
),
leading to formation of nitrosoglutathione (GSNO), which in turn has been
shown to enhance expression of the genes involved in differentiation of brown
adipocytes (Nisoli et al.,
1998
). Moreover, Gaudiot et al.
(1998
,
2000
) showed that GSNO acting
as a NO donor increases basal lipolysis in white fat cells, thus increasing
the concentration of free fatty acids, which are the main fuel for IBAT
thermogenesis (Trayhurn, 1979
;
Bukowiecki et al., 1981
) and
are also known to activate UCP1
(Skulachev, 1991
;
Winkler and Klingerbeg, 1994
).
Since NO action on gene expression has been well documented
(Peunova and Enikolopov, 1995
;
Hobbs, 1997
), it may be
assumed that NO induces the UCP1 gene immediately. However, the
interrelationship of the UCP1 level and NO, i.e. the ability of NO to
stimulate UCP1 expression in IBAT mitochondria, requires further study.
We have also shown that long-term exposure of rats to cold diminished iNOS
level, as well as iNOS immunopositivity, accompanied by a rapid decrease in
the rate of apoptosis in IBAT. The results of Lindquist and Rehnmark
(1998) together with our
recent data (Kora
et al.,
2000
) demonstrated decreased apoptosis in cold-adapted rats and a
significant increase of this process during re-acclimation. Thus, we could
hypothesize that the regulation of cell survival is a process involved in
cold-induced IBAT hyperplasia, i.e. a decreased extent of apoptosis, observed
in cold-maintained rats, could be an additional adaptive change that
contributes to enhanced thermogenic capacity. Changes in both iNOS expression
and intensity of apoptosis in IBAT were parallel in rats kept at room
temperature. In these animals, a marked increase in both the iNOS level and
immunopositivity and the rate of apoptosis were detected in both
L-Arg- and L-NAME-treated rats compared to the
corresponding control. Thus, we suggest that iNOS may be involved in induction
of apoptosis in IBAT.
Generally, NO can induce cell death by apoptosis in a variety of different
cell types (López-Farré et
al., 1996; Messmer et al.,
1996
). There are various biochemical and cellular mechanisms
underlying NO-induced apoptosis, e.g. by inhibiting cytochrome oxidase
(Brown, 2001
;
Cleeterr et al., 1994
),
stimulating production of reactive oxygen species (ROS) and reactive nitrogen
species (RNS; Brown and Borutaite,
2002
), peroxynitrite (ONOO-) formation
(Foresti and Motterlini,
1999
). Also, changes in NO concentration in cells act by shifting
cellular redox potential and turning on or off redox-sensitive genes involved
in proapoptotic/antiapoptotic signal pathways, respectively (Kröncke et
al., 2001). However, further studies along these lines are necessary to
determine possible role of NO in inducing of apoptosis in IBAT.
Our results presented here suggest that NO produced by iNOS could also contribute to overall NO-associated regulation of thermogenesis in IBAT, by enhancing the activity and capacity of IBAT for non-shivering thermogenesis by increasing both the IBAT mass and UCP1 level. Moreover, the cooperative action of decreased apoptosis accompanied by increased tissue hyperplasia and UCP1 level, observed in IBAT of cold-acclimated rats, would be a way to meet the metabolic requirements for increased thermogenesis.
List of abbreviations
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Acknowledgments |
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