Laboratoire de Pharmacologie Médicale et Clinique, Institut National de la Santé et de la Recherche Médicale Unité 317, 31073 Toulouse Cedex, France
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ABSTRACT |
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Uncoupling protein-2 (UCP-2) is a
mitochondrial protein expressed in adipocytes and has recently been
involved in the control of energy dissipation. Because obesity is
characterized by an imbalance between energy intake and expenditure and
by an enhanced adipocyte-derived secretion of tumor necrosis factor-
(TNF-
), we asked whether TNF-
could directly influence UCP-2
expression in adipocytes. Experiments performed in differentiated
3T3F442A preadipocytes showed that TNF-
(10 ng/ml) induced a
reduction of UCP-2 trancripts, assessed by Northern blot analysis. A
significant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF-
stimulation of the cells. The characterization
of the mechanisms responsible for the TNF-
effect on UCP-2
expression demonstrates an involvement of the TNF-
-induced inducible
(i) nitric oxide synthase (NOS) expression. Cell treatment with the NOS
inhibitor NG-nitro-L-arginine methyl
ester (L-NAME; 1 mmol/l) significantly diminished the
TNF-
-mediated sustained downregulation of UCP-2 expression, whereas
cell treatment with a nitric oxide (NO) donor (10
3 mol/l
S-nitroso-L-glutathione) mimicked the TNF-
effect on UCP-2 expression. Moreover, Western blot analysis clearly
showed that TNF-
alone induces the expression of iNOS after
12-24 h treatment of differentiated 3T3F442A cells. These
experiments demonstrate that TNF-
directly downregulates UCP-2
expression via NO-dependent pathways that involve the induction of iNOS expression.
inducible nitric oxide synthase; white fat cells; nitric oxide donor; uncoupling protein-2
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INTRODUCTION |
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ADIPOCYTES ARE ONE OF THE major cellular sites for energy storage in the body through lipogenic and lipolytic regulation processes. They also play an important role in energy homeostasis by modulating thermogenesis. Indeed, adipocytes from brown adipose tissue, mainly developed in rodents, express the mitochondrial uncoupling protein (UCP)-1. UCP-1 is involved in heat production by uncoupling electron transport from the mitochondrial respiratory chain (28). Recently, additional UCP-related genes have been identified. UCP-2 was found to be expressed in a wide range of cells and tissues, including white adipocytes, whereas UCP-3 expression was restricted mainly to skeletal and cardiac muscle (3, 11, 14, 29). Although the function of both UCP-2 and -3 proteins has to be clearly established, several lines of evidence strongly suggest their involvement in the control of energy dissipation. For example, ectopic expression of UCP-2 and -3 in yeast has been shown to decrease the mitochondrial membrane potential associated with uncoupling of respiration (14, 15).
Obesity is characterized by an imbalance between energy intake and
expenditure. Alterations in the control of adipocyte lipogenic and/or
lipolytic processes have been well documented in obesity and revealed,
in most white adipose tissues (WAT) of obese people, a decreased
catecholamine-induced lipolysis. The recent demonstration of a reduced
expression of UCP-2 gene in WAT of obese people (26) has
stressed the new concept of a potential impairment of adipocyte energy
dissipation in obesity. The factors controlling UCP-2 expression in
adipocytes remain to be well characterized. Changes in metabolism such
as fat diet (24) and short-term fasting (2)
have been shown to influence adipocyte UCP-2 expression and various
adipocyte-derived cytokines. Among them, tumor necrosis factor-
(TNF-
), which is produced in high amounts in human obesity
(16, 18), has been recently described to decrease the
expression of UCP-2 in human explants of adipose tissue
(30). However, the mechanism of action of TNF-
involved
in the alteration of UCP-2 expression in adipocytes has not been
studied yet. TNF-
is known to induce the expression of the inducible
(i) nitric oxide synthase (NOS) in various cell types and tissues
(12). The presence of iNOS protein has been demonstrated
recently in adipocytes from rodents (27) and humans
(1) and in 3T3-L1 differentiated adipocytes treated with a
mixture of various cytokines and growth factors (17). In
the present study, we examined the effect of TNF-
on UCP-2
expression in differentiated 3T3F442A adipocytes, and we tested the
hypothesis that the induction of iNOS could be involved in the control
of UCP-2 expression by TNF-
.
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MATERIALS AND METHODS |
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Cell culture.
3T3F442A preadipocyte cells were grown as previously described
(4). Cells were cultured until confluence in a culture
medium consisting of DMEM supplemented with 10% donor calf serum and an antibiotic mixture (500 U/ml penicillin and 50 µg/ml streptomycin) in an atmosphere of 95% air-5% CO2 at 37°C. At
confluence, cells were differentiated by incubation in DMEM
supplemented with 10% FCS and 50 nmol/l insulin. Ten days after
confluence, cells were maintained for 12 h in a serum-deprived
medium containing 0.1% BSA and were treated as described in
RESULTS. After treatment, supernatants were collected for
nitrite determination, and cells were stored at 80°C until analysis.
Measurements of nitrite production. Nitrite concentration was determined in cell supernatant by the Griess reagent [5.44% sulfanylamide; 0.5 mol/l HCl and 0.128% N-(1-naphthyl)ethylenediamine; or 0.5 mol/l HCl (1:1)]. After 10 min at room temperature, absorbance was read at 540 nm (iEMS Labsystem). The nitrite concentrations were calculated from a standard curve obtained with increasing concentrations of NaNO2 (0-50 µmol/l).
Western blot analysis. Cells were washed two times with PBS and scraped. After brief centrifugation (1,000 g, 2 min, 4°C), pellets were resuspended in 200 µl of lysis buffer containing 10 mmol/l Tris · HCl (pH 7.5), 0.15 mol/l NaCl, 2 mmol/l sodium vanadate, 0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mmol/l phenylmethylsulfonyl fluoride, and a mix of protease inhibitors. The lysate was then centrifuged at 13,000 g for 30 min at 4°C, and protein concentrations of the supernatant were determined using a protein determination kit. Proteins (70 µg) were separated by electrophoresis on 10% SDS-8% PAGE under denaturing conditions. After transfer to nitrocellulose membranes and Ponceau staining to verify equal loading of the lanes, membranes were blocked overnight in 50 mmol/l Tris · HCl, 200 mmol/l NaCl, 0.05% Tween 20, and 5% fat milk at 4°C and then were incubated with the primary antibody (mouse anti-iNOS diluted 1:5,000) for 90 min followed by incubation with the secondary antibody (anti-mouse immunoglobulin conjugated with horseradish peroxidase diluted 1:7,500) for 60 min. The immunocomplexes were detected using a chemiluminescence reagent kit.
Extraction of RNA and Northern blot analysis.
Total RNAs were extracted by the standard acid
guanidium-phenol-chloroform method of Chomczynsky and Sacchi
(6) and were dissolved in a solution containing 50%
formamide, 3 mmol/l sodium acetate, 1 mmol/l EDTA, and 2.2 mol/l
formaldehyde. Total RNA samples (20 µg) were heated at 65°C for 10 min, resuspended, and loaded on 1% denaturated agarose gel. After
electrophoresis, RNAs were transferred to a nylon membrane for 1 h
under vacuum (40 mbars for 90 min). The membrane was
prehybridized for 1 h at 65°C in a buffer containing 0.5 mol/l
NaHPO4, 1 mol/l EDTA, 7% SDS, and 1% BSA and was
hybridized overnight with [32P]UCP-2 probe at 65°C in
the same buffer. Membranes were then washed two times in a twofold
concentrated standard saline citrate (SSC; 0.15 mol/l NaCl and 15 mmol/l sodium citrate, pH 7.0) supplemented with 0.1% SDS at room
temperature, two times in 0.2-fold concentrated SSC supplemented with
0.1% SDS at 50°C, and one time in 0.1-fold concentrated SSC
supplemented with 0.1% SDS at 65°C. To confirm equal loading of the
lanes, membranes were stripped and hybridized with a
32P-labeled -actin probe. After 24-h autoradiography at
80°C, the intensity of each band was quantified as integrated areas by using computerized densitometry (Molecular Dynamics, Sunnyvale, CA)
and ImageQuant NT Software. Densitometric values were determined in
areas of equal size and reported in arbitrary units above background values. The intensity of UCP-2 mRNA band was normalized for the
-actin signal in each lane.
Materials.
Chemicals were purchased from Sigma (St. Louis, MO). DMEM and donor
serum and FCS were obtained from Life Technology (Gaithersburg, MD).
S-nitroso-L-glutathione (GSNO) was from Alexis
Biochemicals. Mouse recombinant TNF- was obtained from Sigma
[lipopolysaccharide (LPS) <0.1 ng/µg TNF-
as provided by the
manufacturer]. Preliminary experiments were performed using mouse
recombinant TNF-
(LPS level <0.1 ng/µg TNF-
or 1 endotoxin
unit/µg as provided by the manufacturer) purchased by
PeproTech (TEBU). Mouse anti-iNOS antibody was purchased from
Transduction Laboratories (Lexington, KY), and anti-mouse IgG
conjugated with horseradish peroxidase was from Calbiochem (La Jolla,
CA). LPS from Salmonella typhimurium was obtained from
Sigma. Plasmid containing the mouse UCP-2 cDNA was a generous gift from
Dr. Daniel Riquier (Ceremod, Meudon, France). The radiolabeled
EcoR I/Sac I 935 bp of the UCP-2 cDNA was used as
a probe in the Northern blot experiments. Positive control for iNOS was
prepared from the macrophage cell line RAW 264.7 (provided by Dr.
Bernard Pipy) stimulated by TNF-
(10 ng/ml), interferon-
(IFN-
; 10 ng/ml), and LPS from Escherichia coli (5 µg/ml). The mix of proteases inhibitors was Complete mini tablets from Roche Diagnostics (Meylan, France). Protein concentrations were
assessed by the Bio-Rad DC Protein kit (Bio-Rad, Ivry/Seine, France).
Nitrocellulose and nylon membranes for Western and Northern blot
analysis were purchased from Schleicher & Schuell (Dassel, Germany).
The chemiluminescence reagent kit was the enhanced chemiluminescence kit from Amersham Life Science (Les Ulis, France).
Statistical analysis. Values are expressed as means ± SE. Data were analyzed by an ANOVA followed by a Dunnett's multiple-comparison post hoc test.
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RESULTS |
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Effect of TNF- on UCP-2 mRNA expression.
Northern blot analyses were performed on total RNA extracts obtained
from 10-day differentiated 3T3F442A adipocytes treated for 2, 8, 12, or
24 h with 10 ng/ml mouse recombinant TNF-
. UCP-2 mRNA was
normalized to
-actin expression.
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Induction of iNOS by TNF-.
Because the modulatory effect of TNF-
on UCP-2 expression was only
observed after a 12-h treatment period, we next investigated the
potential involvement of intermediary components in the
TNF-
-dependent pathway involved in the control of UCP-2 expression.
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Involvement of nitric oxide in the TNF--induced downregulation
of UCP-2 expression.
To determine whether the TNF-
-induced iNOS expression and its
concomitant nitric oxide (NO) increase was involved in the TNF-
-mediated downregulation of UCP-2 expression, we analyzed the
effect of the specific NOS inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME) on the TNF-
-induced decrease of UCP-2 mRNA levels.
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DISCUSSION |
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In the present study, we demonstrate that UCP-2 expression in
3T3F442A differentiated adipocytes is downregulated by TNF- treatment. The further analysis of the TNF-
effect on differentiated 3T3F442A adipocytes showed that TNF-
treatment alone induced the
expression of the iNOS protein in a time- and concentration-dependent manner. Inhibition of iNOS activity led to the suppression of TNF-
-mediated downregulation of UCP-2 expression, whereas cell treatment with NO donors led to a reduction of UCP-2 mRNA amounts. Thus
an NO-dependent pathway is involved in the TNF-
-induced downregulation of UCP-2 expression in adipocytes.
UCP-2 belongs to the family of uncoupling proteins recently characterized by the identification and cloning of distinct genes that share homology in their sequences and organization (11, 29). UCP-2 expression has been found in most tissues. However, UCP-2 roles and functions are still not well defined. UCP-2 has been described to uncouple the mitochondrial respiration (11, 14) and thereby might represent a cellular sensor to monitor the efficiency of the system responsible for supplying energy to the cells. Indeed, a positive correlation was found between the resting metabolic rate (minimal energy expenditure required to maintain physiological tissue function) and UCP-2 expression in obese women, suggesting a potential involvement of UCP-2 in the control of energy expenditure (2). Moreover, because its expression has been demonstrated to be modulated in WAT and liver of obese mice, it appears that UCP-2 expression is dependent on metabolic changes (23).
Obesity, characterized by an imbalance between energy intake and energy
expenditure, is associated with enhanced production and secretion by
adipocytes of various cytokines and growth factors, such as leptin and
TNF- (20, 25). Both have been involved in the control
of UCP-2 expression. Leptin cDNA transfected in rats by the use of
adenovirus has been shown to be associated with an increased UCP-2
expression in the WAT (32), whereas direct leptin
administration in mice led to a downregulation of UCP-2 expression
(7). Concerning the effect of TNF-
on UCP-2 expression,
hepatocyte, muscular, and adipocyte UCP-2 trancripts have been shown to
be increased under TNF-
administration in rats or mice (5, 8,
21, 22), whereas in human adipocytes maintained in survival,
TNF-
treatment was reported to lead to a decrease in UCP-2 mRNA
levels (30). Those conflicting data might be explained by
the broad interactions with other TNF-
-dependent pathways when
administrating the cytokine "in vivo" compared with direct "in
vitro" studies. In the present report, we demonstrate that, in
differentiated 3T3F442A adipocytes, TNF-
treatment led to a
downregulation of UCP-2 expression, as assessed by Northern blot
analysis, since a reliable specific antibody is not still available to
perform Western blot analysis.
The TNF--mediated downregulation of UCP-2 transcripts takes place
within 12-24 h. The cytokine-induced sustained effects on gene
expression are generally mediated through the production of
intermediary components. TNF-
is known in various cell types to
increase the production of reactive radicals (10). Indeed, reactive oxygen species (hydrogen peroxide and superoxide anions) have
been described to act as second messengers in TNF-
-mediated regulation of various genes. For example, in rat hepatocytes, reactive
oxygen species have been involved in the TNF-
-induced upregulation
of UCP-2 transcripts (21). Another highly reactive radical, NO, can also be produced under TNF-
stimulation, through iNOS induction (19). We demonstrate, by the use of Western
blot analysis and nitrite determination, that TNF-
stimulation of 3T3F442A differentiated adipocytes is associated with an induction of
iNOS expression and activity. The presence of iNOS has already been
described in WAT from LPS-treated rats (27) and in vivo in
human WAT (1). Moreover, experiments performed in vitro on
the preadipocyte cell line 3T3-L1 have recently demonstrated that iNOS
expression is induced under treatment with a combination of various
cytokines (IFN-
, TNF-
, and LPS; see Ref. 17). We report here
that, in the 3T3F442A preadipocyte cell line, TNF-
alone induces the
iNOS protein and activity. Moreover, the presence of LPS or other
cytokines (IFN-
and interleukin-
) together with TNF-
does not
modify the TNF-
stimulatory effect on iNOS induction (A. Bouloumié, personal communication). This discrepancy between both
cell lines is not explained and requires further investigations. However, because the 3T3F442A cell line is considered in a more advanced differentiation stage compared with the 3T3-L1 cell line, i.e., it requires only insulin addition for differentiation, whereas corticoids, insulin, together with phosphodiesterase inhibitors are
needed for 3T3-L1 cells to differentiate, it can be hypothesized that the 3T3F442A cell line already expresses all the cellular components necessary for the TNF-
-dependent signaling.
The appearance of iNOS protein and nitrite in the cell supernatants
coincides with the second phase of TNF--mediated downregulation of
UCP-2 expression. Furthermore, the inhibition of iNOS activity in the
presence of the NOS inhibitor L-NAME led to an abolition of
the 12-h TNF-
effect on UCP-2 expression. All of those results support the hypothesis that iNOS activity is involved in the long-term effect of TNF-
on UCP-2 expression. To clearly state the role of the
iNOS product NO on the control of UCP-2 expression, cells were treated
with the NO donor GSNO. A downregulation of UCP-2 transcript amounts
was observed in the presence of the donor, further demonstrating the
involvement of NO in the TNF-
-mediated decrease of UCP-2 expression.
The role of NO in adipocytes is not known. Few reports have involved
this radical production in the control of lipolysis (13).
The present report demonstrates a direct effect of NO on the control of
gene expression. The characterization of the molecular pathway
responsible for the NO-mediated decrease of adipocyte UCP-2 expression
remains to be established. On other cell types, NO has been involved in
the modulation of various intracellular transduction pathways,
including modulation regulation of activator protein (AP)-1 and nuclear
factor-
B DNA-binding activities (9). The analysis of
the murine UCP-2 promoter regions has revealed several AP-1 binding
sites and cAMP-response elements that might be potential targets for
the NO-mediated effect on UCP-2 expression in adipocytes
(31).
In conclusion, the present study demonstrates that TNF- decreases
UCP-2 expression in 3T3F442A adipocytes. NO-dependent pathways are
involved in the sustained effect of TNF-
on UCP-2 transcripts. Although further analyses are required to characterize this effect and
to demonstrate its occurrence in vivo, it is tempting to speculate that
the increased production of adipocyte TNF-
observed during obesity
(16, 18) might play a role in the reduction of adipocyte UCP-2 expression and thus contributes to the metabolic imbalance. In
addition, the involvement of NO-dependent pathways in the control of
gene expression in adipocytes opens new fields of investigations concerning the effect of such radical production on adipocyte metabolism.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Institut de Recherche International Servier.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Galitzky, Laboratoire de Pharmacologie Médicale et Clinique, INSERM U317, 31073 Toulouse Cedex, France (E-mail: galitzky{at}cict.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 October 1999; accepted in final form 17 April 2000.
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