Nitric oxide-dependent downregulation of adipocyte UCP-2 expression by tumor necrosis factor-alpha

Christelle Merial, Anne Bouloumie, Véronique Trocheris, Max Lafontan, and Jean Galitzky

Laboratoire de Pharmacologie Médicale et Clinique, Institut National de la Santé et de la Recherche Médicale Unité 317, 31073 Toulouse Cedex, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ), we asked whether TNF-alpha could directly influence UCP-2 expression in adipocytes. Experiments performed in differentiated 3T3F442A preadipocytes showed that TNF-alpha (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-alpha stimulation of the cells. The characterization of the mechanisms responsible for the TNF-alpha effect on UCP-2 expression demonstrates an involvement of the TNF-alpha -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-alpha -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-alpha effect on UCP-2 expression. Moreover, Western blot analysis clearly showed that TNF-alpha alone induces the expression of iNOS after 12-24 h treatment of differentiated 3T3F442A cells. These experiments demonstrate that TNF-alpha 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ), 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-alpha involved in the alteration of UCP-2 expression in adipocytes has not been studied yet. TNF-alpha 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-alpha 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-alpha .


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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-alpha was obtained from Sigma [lipopolysaccharide (LPS) <0.1 ng/µg TNF-alpha as provided by the manufacturer]. Preliminary experiments were performed using mouse recombinant TNF-alpha (LPS level <0.1 ng/µg TNF-alpha 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-alpha (10 ng/ml), interferon-gamma (IFN-gamma ; 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of TNF-alpha 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-alpha . UCP-2 mRNA was normalized to beta -actin expression.

As shown in Fig. 1, short-term TNF-alpha stimulation on differentiated 3T3F442A cells (from 2 to 8 h) had no significant effect on UCP-2 transcript levels compared with unstimulated cells. TNF-alpha treatment for 12 h led to a significant decrease in UCP-2 expression (40% decrease in UCP-2 mRNA amount in TNF-alpha -treated cells compared with control cells, P < 0.01, n = 3). The TNF-alpha -mediated downregulation of UCP-2 mRNA expression remained for the next 12-h treatment (P < 0.01, n = 3)


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of tumor necrosis factor-alpha (TNF-alpha ) treatment on uncoupling protein (UCP)-2 expression. 3T3F442A differentiated adipocytes were treated (T) or not (C) for 2, 8, 12, or 24 h with TNF-alpha (10 ng/ml). RNA (20 µg/lane) was separated by electrophoresis, transferred to membrane, and hybridized with [32P]UCP-2 or beta -actin cDNA. A: representative autoradiography from Northern blot analysis. B: intensity of UCP-2 mRNA band was normalized for the beta -actin signal in each lane by densitometric analysis. Data are expressed as percentage of control and are means ± SE from 3 independent experiments. ** P < 0.01 vs. time 0.

Induction of iNOS by TNF-alpha . Because the modulatory effect of TNF-alpha 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-alpha -dependent pathway involved in the control of UCP-2 expression.

Western blot analysis, performed on crude protein extracts obtained from 10 day-differentiated 3T3F442A cells treated for increasing time with 10 ng/ml mouse recombinant TNF-alpha revealed the time-dependent appearance of a 130-kDa band identified with the antibody directed against iNOS (Fig. 2A). The 130-kDa band was not detectable in control cells at any time point or in cells treated with TNF-alpha for 2-8 h. However, iNOS protein was evidenced after 12 h of TNF-alpha stimulation, and iNOS expression increased during the next 12-h treatment with TNF-alpha . In parallel, nitrite determination, performed on cell supernatants, demonstrated a significant accumulation of nitrites after a 12-h stimulation with TNF-alpha (1.8-fold increase in TNF-alpha -treated cells compared with untreated cells, P < 0.05, n = 3; Fig. 2B). The nitrite accumulation in cell supernatant under TNF-alpha treatment was further enhanced after 24 h (4.8-fold increase in TNF-alpha -treated cells compared with untreated cells, P < 0.01, n = 3), demonstrating the presence of a functional iNOS under TNF-alpha stimulation of 3T3F442A differentiated adipocytes.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of TNF-alpha on inducible (i) nitric oxide synthase (NOS) expression and nitrite release. 3T3F442A differentiated adipocytes were treated with TNF-alpha (10 ng/ml) during 2, 8, 12, or 24 h. A: protein extracts were analyzed by Western blot using a specific iNOS antibody. A representative autoradiography of Western blot analysis from 3 independent experiments is shown. Cont, control. B: nitrite (NO2) concentrations in cell supernatant were determined using the Griess reagent. Results are expressed as means ± SE from 3 independent experiments. * P < 0.05 and ** P < 0.01 vs. control.

To further characterize the direct effect of TNF-alpha on iNOS induction in adipocytes, we studied the TNF-alpha concentration dependency on iNOS induction. Western blot analysis performed on crude protein extracts obtained from 10-day differentiated 3T3F442A cells treated for 24 h with increasing concentrations of TNF-alpha (0.5-50 ng/ml) showed a concentration-dependent induction of iNOS expression. The iNOS protein was detected at a concentration as low as 2.5 ng/ml TNF-alpha and was maximal at the concentration of 20 ng/ml (Fig. 3A). In parallel, a TNF-alpha concentration-dependent increase in nitrite detected in cell supernatant (Fig. 3B) was observed, further demonstrating that TNF-alpha alone is able to induce iNOS expression in adipocytes. This effect was specific to TNF-alpha , since LPS alone did not induce iNOS expression and nitrite accumulation (2.8 ± 0.4 µmol/l nitrites in 10 µg/ml LPS-treated cells vs. 2.5 ± 0.5 µmol/l nitrites in control cells) and did not modify the TNF-alpha stimulatory effect (14 ± 2 µmol/l nitrites in 10 µg/ml LPS together with 10 ng/ml TNF-alpha -treated cells vs. 17 ± 3 µmol/l nitrites in 10 ng/ml TNF-alpha -treated cells). Moreover, preliminary experiments performed with other TNF-alpha sources gave identical results (7.1-fold increase in nitrite accumulation in 10 ng TNF-alpha -treated cells vs. control cells, n = 5, P < 0.01), showing then that TNF-alpha alone is responsible for the iNOS induction.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of TNF-alpha on iNOS expression. Differentiated adipocytes were treated with increasing concentrations of TNF-alpha (0, 0.5, 1, 2.5, 5, 10, 20, or 50 ng/ml). A: protein extracts were analyzed by Western blot using a specific iNOS antibody. A representative autoradiography of Western blot analysis from 3 independent experiments is shown. B: nitrite concentrations were assessed in cell supernatants using the Griess reagent. Results are expressed as means ± SE from 3 independent experiments. ** P < 0.01 vs. 0 ng/ml TNF-alpha .

Involvement of nitric oxide in the TNF-alpha -induced downregulation of UCP-2 expression. To determine whether the TNF-alpha -induced iNOS expression and its concomitant nitric oxide (NO) increase was involved in the TNF-alpha -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-alpha -induced decrease of UCP-2 mRNA levels.

L-NAME alone (1 mmol/l) for 12 h did not affect UCP-2 expression, as assessed by Northern blot analysis (Fig. 4A). However, the downregulation in UCP-2 mRNA amount observed under TNF-alpha treatment was suppressed in the presence of L-NAME (Fig. 4A). UCP-2 mRNA levels in cells treated for 12 h with both TNF-alpha and L-NAME were not significantly different compared with control cells (n = 3). In parallel, L-NAME led to a complete inhibition of iNOS activity observed under TNF-alpha treatment, since no nitrite accumulation could be detected in supernatants from cells treated for 12 h with TNF-alpha and L-NAME (Fig. 4B). The effect of L-NAME was specific since its inactive enantiomer D-NAME had no effect on the TNF-alpha -induced downregulation of UCP-2 expression (data not shown).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME)] on TNF-alpha -induced decrease of UCP-2 expression. Cells were treated or not (C) with TNF-alpha (10 ng/ml), L-NAME (LN, 1 mmol/l), or TNF-alpha  + L-NAME (TNFalpha  + LN) for 12 h. A: Northern blot analyses were performed on RNA extracts, and representative autoradiographs are shown. The intensity of UCP-2 mRNA band was normalized for the beta -actin signal in each lane by densitometric analysis. Data are expressed as percentage of control and are means ± SE from 3 independent experiments. ** P < 0.01 vs. control. B: nitrite concentrations were assessed in cell supernatants using the Griess reagent. Results are expressed as means ± SE from 3 independent experiments. * P < 0.05 vs. control cells.

Finally, we studied the effect of an NO donor on UCP-2 expression. Differentiated 3T3F442A adipocytes were treated for 4 h with GSNO at increasing concentrations (0.01, 0.1, and 1 mmol/l). Northern blot analysis revealed that GSNO induced a significant decrease in UCP-2 transcripts in a concentration-dependent manner (Fig. 5). A 40% decrease in UCP-2 mRNA levels was observed with the concentration of 1 mmol/l compared with untreated cells (P < 0.05, n = 3). The GSNO-induced downregulation of UCP-2 expression was still present after 12-24 h (data not shown).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of NO donor [S-nitroso-L-glutathione (GSNO)] on UCP-2 expression. Cells were treated for 4 h with increasing concentrations of GSNO (0.01, 0.1, and 1 mmol/l). Northern blot analyses were performed on RNA extracts, and representative autoradiographs are shown. The intensity of UCP-2 mRNA band was normalized for the beta -actin signal in each lane by densitometric analysis. Data are expressed as percentage of control and are means ± SE from 3 independent experiments. * P < 0.05 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrate that UCP-2 expression in 3T3F442A differentiated adipocytes is downregulated by TNF-alpha treatment. The further analysis of the TNF-alpha effect on differentiated 3T3F442A adipocytes showed that TNF-alpha 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-alpha -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-alpha -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-alpha (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-alpha on UCP-2 expression, hepatocyte, muscular, and adipocyte UCP-2 trancripts have been shown to be increased under TNF-alpha administration in rats or mice (5, 8, 21, 22), whereas in human adipocytes maintained in survival, TNF-alpha 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-alpha -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-alpha 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-alpha -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-alpha 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-alpha -mediated regulation of various genes. For example, in rat hepatocytes, reactive oxygen species have been involved in the TNF-alpha -induced upregulation of UCP-2 transcripts (21). Another highly reactive radical, NO, can also be produced under TNF-alpha stimulation, through iNOS induction (19). We demonstrate, by the use of Western blot analysis and nitrite determination, that TNF-alpha 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-gamma , TNF-alpha , and LPS; see Ref. 17). We report here that, in the 3T3F442A preadipocyte cell line, TNF-alpha alone induces the iNOS protein and activity. Moreover, the presence of LPS or other cytokines (IFN-gamma and interleukin-beta ) together with TNF-alpha does not modify the TNF-alpha 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-alpha -dependent signaling.

The appearance of iNOS protein and nitrite in the cell supernatants coincides with the second phase of TNF-alpha -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-alpha effect on UCP-2 expression. All of those results support the hypothesis that iNOS activity is involved in the long-term effect of TNF-alpha 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-alpha -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-kappa 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-alpha decreases UCP-2 expression in 3T3F442A adipocytes. NO-dependent pathways are involved in the sustained effect of TNF-alpha 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-alpha 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.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Institut de Recherche International Servier.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andersson, K, Gaudiot N, Ribière C, Elizalde M, Giudicelli Y, and Arner P. A nitric oxide-mediated mechanism regulates lipolysis in human adipose tissue in vivo. Br J Pharmacol 126: 1639-1645, 1999[Abstract/Free Full Text].

2.   Barbe, P, Millet L, Larrouy D, Galitzky J, Berlan M, Louvet J, and Langin D. Uncoupling protein-2 messenger ribonucleic acid expression during very-low-calorie diet in obese premenopausal women. J Clin Endocrinol Metab 83: 2450-2453, 1998[Abstract/Free Full Text].

3.   Boss, O, Samec S, Paoloni-Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P, and Giacobino J. Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression. FEBS Lett 408: 39-42, 1997[ISI][Medline].

4.   Bouloumié, A, Planat V, Devedjian J, Valet P, Saulnier-Blache J, Record M, and Lafontan M. Alpha 2-adrenergic stimulation promotes preadipocytes proliferation. Involvement of mitogen-activated protein kinases. J Biol Chem 269: 30254-30259, 1994[Abstract/Free Full Text].

5.   Busquet, S, Sanchis D, Alvarez B, Ricquier D, Lopez-Soriano F, and Argilés J. In the rat, tumor necrosis factor alpha  administration results in an increase in both UCP-2 and UCP-3 mRNAs in skeletal muscle: a possible mechanism for cytokine-induced thermogenegis. FEBS Lett 440: 348-350, 1998[ISI][Medline].

6.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-cloroform extraction. Anal Biochem 162: 156-169, 1987[ISI][Medline].

7.   Combatsiaris, T, and Charron M. Downregulation of uncoupling protein-2 mRNA in white adipose tissue and uncoupling protein-3 mRNA in squeletal muscle during the early stages of leptin treatment. Diabetes 48: 128-133, 1999[Abstract].

8.   Cortez-Pinto, H, Yang SQ, Lin HZ, Costa S, Hwang C-S, Lane M, Bagby G, and Diehl A. Bacterial lipopolysaccharide induces uncoupling protein-2 expression in hepatocytes by tumor necrosis factor-alpha -dependent mechanism. Biochem Biophys Res Commun 251: 313-319, 1998[ISI][Medline].

9.   Eder, J. Tumor necrosis factor alpha  and interleukin 1 signalling: MAPKK kinases connect it all? TIPS 18: 319-322, 1997[Medline].

10.   Fiers, W. Tumor necrosis factor. Characterization at the molecular, cellular and in vivo level. FEBS Lett 285: 199-212, 1991[ISI][Medline].

11.   Fleury, C, Neverona M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit R, Ricquier D, and Warden C. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 15: 269-272, 1997[ISI][Medline].

12.   Förstermann, U, Gath I, Schwarz P, Closs E, and Kleinert H. Isoforms of nitric oxide synthase: protperties, cellular distribution and expressional control. Biochem Pharmacol 50: 1321-1332, 1995[ISI][Medline].

13.   Gaudiot, N, Jaubert AM, Charbonnier E, Sabourault D, Lacasa D, Giudicelli Y, and Ribière C. Modulation of white adipose tissue lipolysis by nitric oxide. J Biol Chem 273: 13475-13481, 1998[Abstract/Free Full Text].

14.   Gimeno, R, Dembski M, Weng X, Deng N, Shyjan A, Gimeno C, Iris F, Ellis S, Woolf E, and Tartaglia L. Cloning and characterization of an uncoupling protein homolog. A potential modulator of human thermogenesis. Diabetes 46: 900-906, 1997[Abstract].

15.   Gong, D, He Y, Karas M, and Reitman M. Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists and leptin. J Biol Chem 272: 24129-24132, 1997[Abstract/Free Full Text].

16.   Hostamisligil, G, Arner P, Caro J, Atkinson R, and Speigelman B. Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin-resistance. J Clin Invest 95: 2409-24015, 1995[ISI][Medline].

17.   Kapur, S, Marcotte B, and Marette A. Mechanism of adipose tissue iNOS induction in endotoxemia. Am J Physiol Endocrinol Metab 276: E635-E641, 1999[Abstract/Free Full Text].

18.   Kern, P, Saghizadeh M, Ong J, Bosch R, Deem R, and Simsolo R. The expression of tumor necrosis factor in human adipose tissue. J Clin Invest 95: 2111-2119, 1995[ISI][Medline].

19.   Kilbourn, R, Gross S, Jubran A, Adams J, Griffith O, Levi R, and Lodato R. NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide. Proc Natl Acad Sci USA 87: 3629-3632, 1990[Abstract].

20.   Kirchgessner, T, Uysal K, Wiesbrock S, Marino M, and Hostamisligil G. Tumor necrosis factor-alpha contributes to obesity-related hyperleptinemia by regulating leptin release from adipocytes. J Clin Invest 100: 2777-2782, 1997[Abstract/Free Full Text].

21.   Lee, F, Li Y, Zhu H, Yang S, Lin H, Trush M, and Diehl A. Tumor necrosis factor increases mitochondrial oxidant production and induces expression of uncoupling protein-2 in the regenerating mice liver. Hepatology 29: 677-687, 1999[ISI][Medline].

22.   Masaki, T, Yoshimatsu H, Chiba S, Hidaka S, Tajima D, Kakuma T, Kurokawa M, and Sakata T. Tumor necrosis factor-alpha regulates in vivo expression of the rat UCP family differentially. Biochim Biophys Acta 1436: 585-592, 1999[ISI][Medline].

23.   Matsuda, J, Hosoda K, Itoh H, Son C, Doi K, Tanaka T, Fukunaga Y, Inoue G, Nishimura H, Yoshimasa Y, Yamori Y, and Nakao K. Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet. FEBS Lett 418: 200-204, 1997[ISI][Medline].

24.   Millet, L, Vidal H, Andrealli F, Larrouy D, Riou J, Riquier D, Laville M, and Langin D. Increased uncoupling protein-2 and uncoupling protein-3 mRNA expression during fasting in obese and lean humans. J Clin Invest 100: 2665-2670, 1997[Abstract/Free Full Text].

25.   Mohamed-Ali, V, Pinkney J, and Coppack S. Adipose tissue as an endocrine and paracrine organ. Int J Obes 22: 1145-1158, 1998[ISI][Medline].

26.   Oberkofler, H, Liu Y, Esterbauer H, Hell E, Kempler F, and Patsch W. Uncoupling protein-2 gene: reduced mRNA expression in intraperitoneal adipose tissue of obese humans. Biochem Biophys Res Commun 41: 940-946, 1998.

27.   Ribière, C, Jaubert A-M, Gaudiot N, Sabourault D, Marcus M, Boucher J, Denis-Henriot D, and Giudicelli Y. White adipose tissue nitric oxide synthase: a potential source for NO production. Biochem Biophys Res Commun 222: 706-712, 1996[ISI][Medline].

28.   Ricquier, D, Casteilla L, and Bouillaud F. Molecular studies of the uncoupling protein. FASEB J 5: 2237-2242, 1991[Abstract/Free Full Text].

29.   Vidal-Puig, A, Sloanes G, Grujic D, Flier J, and Lowell B. UCP-3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue. Biochem Biophys Res Commun 235: 79-82, 1997[ISI][Medline].

30.   Viguerie-Bascands, N, Saulnier-Blache JS, Dandine M, Dauzats M, Daviaud D, and Langin D. Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes. Biochem Biophys Res Commun 256: 138-141, 1999[ISI][Medline].

31.   Yoshitomi, H, Yamazaki K, and Tanaka I. Mechanism of ubiquitous expression of mouse uncoupling protein-2 mRNA: control by cis-acting DNA element in 5'-flancking region. Biochem J 340: 397-404, 1999[ISI][Medline].

32.   Zhou, Y-T, Shimabukuro M, Koyama K, Lee Y, Wang M-Y, Trieu F, Newgard C, and Unger R. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation. Proc Natl Acad Sci USA 94: 6386-6390, 1997[Abstract/Free Full Text].


Am J Physiol Cell Physiol 279(4):C1100-C1106
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (7)
Google Scholar
Articles by Merial, C.
Articles by Galitzky, J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Merial, C.
Articles by Galitzky, J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online