Modulation of White Adipose Tissue Lipolysis by Nitric Oxide*

Nicolas Gaudiot, Anne-Marie Jaubert, Elisabeth Charbonnier, Dominique Sabourault, Danièle Lacasa, Yves GiudicelliDagger , and Catherine Ribière

From the Department of Biochemistry, INSERM CJF 94--02, Faculté de Médecine de Paris-Ouest, Universite René Descartes, Paris, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

In isolated adipocytes, the nitrosothiols S-nitroso-N-acetyl-penicillamine (SNAP) and S-nitrosoglutathione stimulate basal lipolysis, whereas the nitric oxide (NO·) donor 1-propamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA-NONOate) or NO gas have no effect. The increase in basal lipolysis due to nitrosothiols was prevented by dithiothreitol but not by a guanylate cyclase inhibitor. In addition the cyclic GMP-inhibited low Km, cyclic AMP phosphodiesterase activity was inhibited by SNAP suggesting that SNAP acting as NO+ donor increases basal lipolysis through a S-nitrosylation mediated inhibition of phosphodiesterase. Contrasting with these findings, SNAP reduced both isoproterenol-stimulated lipolysis and cyclic AMP production, whereas it failed to modify forskolin-, dibutyryl cyclic AMP-, or isobutylmethylxanthine-stimulated lipolysis, suggesting that SNAP interferes with the beta -adrenergic signal transduction pathway upstream the adenylate cyclase. In contrast with SNAP, PAPA-NONOate or NO gas inhibited stimulated lipolysis whatever the stimulating agents used without altering cyclic AMP production. Moreover PAPA-NONOate slightly reduces (30%) the hormone-sensitive lipase (HSL) activity indicating that stimulated lipolysis inhibition by NO· is linked to both inhibition of the HSL activity and the cyclic AMP-dependent activation of HSL. These data suggest that NO· or related redox species like NO+/NO- are potential regulators of lipolysis through distinct mechanisms.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Nitric oxide (NO)1 has emerged as a chemical messenger in several biological systems. This molecule, which is the smallest biological signal known in mammalian cells, can control vital functions such as neurotransmission and blood vessel tone as well as host defense and immunity (1, 2). Some of the effects due to NO are elicited through the activation of soluble guanylate cyclase, leading to an increase in intracellular cyclic GMP content (3). In addition NO and NO-related species interact with redox metal-containing proteins and/or with thiol groups of proteins (4). It has been suggested that S-nitrosylation of proteins could mediate signaling functions and that thiols may be involved in the stabilization and metabolism of NO (5, 6).

NO is synthesized via L-arginine oxidation by a family of nitric oxide synthase isoforms (NOS). NOS are either constitutively expressed and calcium/calmodulin-dependent (NOS I and NOS III were originally described in neuronal tissue and endothelial cells, respectively) or inducible and almost calcium/calmodulin-independent (NOS II was originally identified in macrophages) (7). We have recently shown that white adipose tissue expresses the NOS II and NOS III isoforms (8). The constitutive expression of NOS II in this tissue can be related to the fact that tumor necrosis factor alpha  is expressed and secreted by adipose tissue, which is also an important target of this cytokine (9). Indeed tumor necrosis factor alpha  was reported to decrease lipoprotein lipase activity and expression (10, 11) and to stimulate lipolysis in white adipocytes (10, 12, 13). However, the role of NO in these cells is so far unknown. Because of the stimulatory effect of tumor necrosis factor alpha  on both NOS II and lipolysis, a role for NO as a putative regulatory signal controlling lipolysis is questionable.

The purpose of this study was to test this hypothesis by investigating the influence of NO itself and different NO donors of various reactive nitrogen intermediates on lipolysis in rat fat cells. The donors tested were (i) two nitrosothiols, S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione (GS-NO) that are described as performing protein S-nitrosylation through the NO+ properties of their NO group (14) and (ii) one compound belonging to the NONOate family, PAPA-NONOate, that generates NO· (15). The rate-limiting step of adipocyte lipolysis is the hydrolysis of triacyl glycerol by the hormone-sensitive lipase (HSL). The main mechanism involves phosphorylation of the HSL by protein kinase A. Therefore, hormones that affect cyclic AMP levels modulate lipolysis. The stimulatory effect of catecholamines on lipolysis is connected to the beta -adrenergic receptor-controlled increment of intracellular cyclic AMP concentrations. In the present study we report the effect of NO and related species on basal lipolysis as well as on stimulated lipolysis by isoproterenol or agents acting on cyclic AMP levels.

Here, we provide clear evidence that NO modulates lipolysis through different mechanisms that appear dependent on the redox forms of NO.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitrosoglutathione (GS-NO), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), 1-propamine,3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA-NONOate) were purchased from Cayman (SPI-Bio, France); 1H(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ) was purchased from Alexis Corp. (Coger, France). Dibutyryl cyclic AMP, forskolin, 3-isobutyl-1-methylxanthine (IBMX), isoproterenol bitartrate salt, bovine serum albumin fraction V (free fatty acid), dithiothreitol (DTT), gum arabic, and snake venom were obtained from Sigma (France). Adenosine deaminase and glycerol kit assay were purchased from Boehringer Mannheim (France). Radiochemical binding assay kit for cyclic AMP (TRK-432), [3H]cyclic adenosine monophosphate NH4 salt, glycerol tri[14C]oleate, [14C]oleic acid were purchased from Amersham International (France). Collagenase type I (267 units/mg) was obtained from Worthington and AG1-X2 resin from Bio-Rad.

Preparation of Isolated Adipocytes-- Male Sprague-Dawley rats (240-250 g), fed ad libitum, were killed by decapitation and epididymal fat pads were removed for adipocyte isolation. According to a modification (16) of the method of Rodbell (17), 1 g of adipose tissue was digested in a plastic vial with 8 ml of Krebs-Ringer-Tris buffer (pH 7.4) (KRT) containing 3% (w/v) bovine serum albumin and 2 mg of collagenase/ml. After 15 min at 37 °C under constant shaking, cells were dispersed and filtered through a nylon mesh and washed four times with KRT containing 1% (w/v) bovine serum albumin.

Lipolysis Studies-- Fat cells (1 × 105-2 × 105) were incubated during 1 h at 37 °C in KRT containing 5 mM glucose, 2% (w/v) bovine serum albumin, 50 milliunits/ml adenosine deaminase in the absence or presence of the effectors to be tested. Lipolysis was stopped by centrifugation (5,000 × g, 15 min at 4 °C), cells were aspirated, and glycerol release was determined with an enzymatic method according to Wieland (18).

Determination of Cyclic AMP-- Fat cells were incubated 15 min at 37 °C in 1 ml of KRT containing 5 mM glucose, 2% (w/v) bovine serum albumin, 50 milliunits/ml adenosine deaminase and 0.1 mM IBMX with or without the different effectors to be tested. Incubations were stopped by adding 5 mM EDTA followed by a 3 min immersion in a boiling-water bath. Cyclic AMP production was determined using a radiochemical binding assay kit (Amersham Pharmacia Biotech). Radioactivity was counted by addition of liquid scintillation mixture (Aqua Safe 300 plus, Zinsser Analytic).

HSL Assay-- HSL activity was performed according to a modification of the procedure of Khoo and Steinberg (19). Fat pads of fasted rats were homogenized in buffer containing 0.25 M sucrose, 1 mM EDTA, and 10 mM Tris/HCl buffer, pH 7.4 (1 g/2.5 vol). The fat cake was discarded by slow centrifugation and the resulting infranatant was recentrifuged at 20,000 × g for 20 min. The supernatant was used for HSL activity determination. The assays were conducted as follows: each vial contained 0.2 ml of enzyme preparation and 0.6 ml of substrate emulsion consisting of glycerol tri[1-14C]oleate (specific activity 50 mCi/mmol) and unlabeled triolein (1.04 µmol), 20 mg of bovine serum albumin, 5 mg of arabic gum, all in 50 mM sodium phosphate buffer, pH 6.8. Incubations were performed for 30 min at 37 °C and released [1-14C]oleic acid was measured by the method of Belfrage and Vaughan (20). Recovery of the extraction procedure was tested with [14C]oleic acid alone and was about 80%. HSL activity was calculated as µmol of free fatty acid released per mg of protein per hour.

Cyclic AMP Phosphodiesterase (PDE) Activity-- Isolated adipocytes were homogenized in buffer containing 20 mM Tris (pH 7.5), 1 mM EDTA, 0.25 M sucrose, 2 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride. The fat cake was removed by centrifugation (3,000 × g). The infranatant was centrifuged at 48,000 × g for 30 min. The pellet was resuspended in 10 mM TES (pH 7.5), 5 mM MgCl2 to yield a final protein concentration of 0.5 mg/ml. 20 µl of the resulting suspension was immediately used for cyclic AMP PDE assays as follows. The low Km cyclic AMP PDE activity was determined at 30 °C during 10 min in the presence of 0.02 µM cyclic [3H]AMP, 0.5 µM cyclic AMP, 10 mM TES buffer (pH 7.5), 5 mM MgCl2, 0.04% (w/v) bovine serum albumin and 0.5 units/ml adenosine deaminase, in a final volume of 100 µl (21). Incubations were stopped by a 1.5-min immersion in a boiling water bath, followed by the addition of 50 µl of 2 mg/ml snake venom in 0.1 M Tris (pH 8) to each tube. After 10 min of incubation at 30 °C, reactions were achieved by a 1.5-min immersion in boiling water. 300 µl of 2:1 (v/v) H2O/AG1-X2 resin were then added to each tube. The tubes were vigorously shaken for 5 min and centrifuged at 3,000 × g for 10 min. 100 µl of the supernatant were removed and counted. Blank values obtained from tubes incubated with boiled cells were substracted.

Preparation of NO Solutions-- NO solutions were prepared by bubbling N2 through KRT buffer to remove O2. NO gas was then bubbled in this buffer for 30 min to saturate the solution. NO concentration was determined by spectrophotometric assay using oxyhemoglobin (22). Aliquots of the NO solution were added to the incubation medium of adipocytes used for the determination of lipolysis.

Other Determinations-- Protein concentrations were determined according to the method of Bradford (23) and cell numbers were calculated according to Hirsh and Gallian (24). All results were expressed as the mean ± S.E. Comparisons between groups were made using paired Student's t test.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Effects of NO Donors on Basal Lipolysis-- To evaluate the effect of NO or NO-related species on basal lipolysis, isolated adipocytes were incubated in the presence of different chemical NO donors. In a dose-dependent manner, the nitrosothiols, SNAP and GS-NO, produced an increase in glycerol release (Fig. 1). This effect was reversed when adipocytes were exposed to the NO scavenger carboxy-PTIO (200 µM) (25) (Fig. 1A), suggesting that the lipolytic effects due to this nitrosothiol are linked to NO generation. In contrast, adipocyte exposure to another NO donor (PAPA-NONOate) or to NO gas (10-500 µM) failed to elicit any significant effect on the basal lipolytic activity (Fig. 2, A and B). Thus NO only generated from nitrosothiols appears to be an activator of basal lipolysis.


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Fig. 1.   Dose-dependent stimulatory effects of nitrosothiols on basal lipolysis. Isolated adipocytes were incubated in Krebs-Ringer-Tris buffer for 1 h with the indicated concentrations of SNAP with or without 200 µM carboxy-PTIO (A) or GS-NO (B). Lipolysis was measured as the amount of glycerol released to the incubation medium. Results are expressed as the percentage of basal lipolysis from nontreated adipocytes (control: A = 39 ± 7; B = 33 ± 9 nmol glycerol/105 cells/h) and are means ± S.E. of separate experiments performed in duplicate. Statistical comparisons were made by the paired Student's t test. * p < 0.01 versus control; dagger  p < 0.01 versus SNAP.


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Fig. 2.   Failure of PAPA-NONOate or authentic NO· to affect basal lipolysis. Isolated adipocytes were incubated in Krebs-Ringer-Tris buffer for 1 h with the indicated concentrations of PAPA-NONOate (A) or NO gas from a saturated buffer solution (B). Lipolysis is expressed as nmol of glycerol/105 cells/h. Results are means ± S.E. of three separate experiments performed in duplicate.

To determine whether cyclic GMP plays any role in the lipolytic response of adipocytes to nitrosothiols, we tested the effect of ODQ, a potent and specific inhibitor of soluble guanylate cyclase (26). As shown in Fig. 3, a 15-min pretreatment of the fat cells by 100 µM ODQ did not prevent the increased lipolysis induced by 0.5-2 mM SNAP, suggesting that the lipolytic effect of nitrosothiols is cyclic GMP-independent.


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Fig. 3.   Lack of effect of the soluble guanylate cyclase inhibitor (ODQ) on SNAP-stimulated lipolysis. Isolated adipocytes were preincubated for 15 min with 100 µM ODQ before the addition of SNAP. Results are expressed as the percentage of basal lipolysis from nontreated adipocytes (control: 27 ± 2 nmol of glycerol/105 cells/h) and are means ± S.E. of four separate experiments performed in duplicate.

The nitrosothiol effect on basal lipolysis could be mediated through direct redox and/or S-nitrosylation reactions at a "redox switch(es)" containing critical thiol groups. To test this hypothesis, we examined the effect of DTT (0.5 mM), a thiol-reducing agent that is also able to modify the redox form of NO (14). Under these conditions, data in Fig. 4 show that the lipolytic effect of SNAP was completely abolished. This finding suggests that the SNAP effects are most probably mediated through interaction with a redox switch involving direct S-nitrosylation and/or disulfide reactions. As nitrosothiols are relatively cell impermeable (27), interaction of SNAP with extracellular or membraneous thiols coud be hypothesized. Generally lipolysis is promoted by a rise in cyclic AMP resulting from adenylate cyclase activation and/or cyclic AMP phosphodiesterase inhibition. Among the cyclic AMP phosphodiesterase isoenzymes, the particulate cyclic GMP-inhibited low Km cyclic AMP phosphodiesterase (cGI-PDE) appears to play a central regulatory role in the control of cyclic AMP level in adipocytes (28-30). Since this enzyme has been reported to be highly sensitive to inhibition by agents that covalently modify protein sulfhydryls (31), the effects of SNAP on adipocyte cGI-PDE activity were investigated. Table I shows that SNAP when used at concentrations activating lipolysis inhibits cGI-PDE activity, contrary to PAPA-NONOate, which was without any effect. These findings led us to study the effect of SNAP on cyclic AMP production in the presence of IBMX, an inhibitor of PDE, to mask the potential inhibitory effect of SNAP on PDE. Under these conditions, we failed to observe any modification of cyclic AMP production in response to 0.5-2 mM of SNAP (data not shown). Altogether, these results indicate that the increased basal lipolytic activity observed with nitrosothiols is likely due to the inhibition of PDE.


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Fig. 4.   Interaction of dithiothreitol with SNAP-stimulated lipolysis. Isolated adipocytes were incubated with the indicated concentrations of SNAP either in the absence or in the presence of 0.5 mM DTT. Results are expressed as the percentage of basal lipolysis from nontreated adipocytes (control: 34 ± 5 of nmol glycerol/105 cells/h) and are means ± S.E. of four separate experiments performed in duplicate. Statistical comparisons were made by the paired Student's t test. * p < 0.01 versus SNAP.

                              
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Table I
SNAP decreases the cyclic GMP-inhibited low Km cyclic AMP phosphodiesterase activity, whereas PAPA-NONOate fails to modify this enzyme activity
Adipocytes were homogenized and cyclic AMP phosphodiesterase activity was measured as described under "Experimental Procedures." Each value obtained in the presence of NO donors is also expressed as percentage of the control and is indicated in parentheses. Results are expressed as mean ± S.E. of three separate experiments performed in duplicate. Statistical analysis was made by paired Student's t test. * p < 0.05, ** p < 0.01 versus control.

Effects of NO Donors and Authentic NO on Stimulated Lipolysis-- Lipolysis was stimulated using different agents acting: (i) at the beta -adrenergic receptor level (isoproterenol); (ii) at the adenylate cyclase level (forskolin); (iii) at the PDE level (isobutylmethylxanthine); (iv) or at the protein kinase level (dibutyryl-cyclic AMP).

Effects of Nitrosothiols-- As shown in Fig. 5 and contrasting with the above described data, SNAP decreased isoproterenol-stimulated lipolysis. Surprisingly, the antilipolytic effect elicited by 2 mM SNAP was not prevented by addition of 0.5 mM DTT or 100 µM ODQ (Fig. 5). Using GS-NO as another NO donor, the same results were observed (data not shown). SNAP also decreased the magnitude of the isoproterenol-stimulated cyclic AMP response (Table II). However, lipolysis stimulated by 1 mM dibutyryl-cyclic AMP, 0.1 mM IBMX or 10 µM forskolin were not significantly modified by SNAP (Fig. 6) as was the cyclic AMP response to 10 µM forskolin (Table II). Taken altogether, these results strongly suggest that the SNAP inhibitory effect toward catecholamine-stimulated lipolysis mainly results from the interaction of SNAP with the beta -adrenergic receptors and/or the Gs protein.


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Fig. 5.   SNAP inhibits isoproterenol-stimulated lipolysis. Isolated adipocytes were incubated in Krebs-Ringer-Tris buffer for 1 h with increasing concentrations of SNAP in the presence of 1 µM isoproterenol. When indicated adipocytes were incubated with 0.5 mM DTT in the presence of 2 mM SNAP or preincubated for 15 min with 100 µM ODQ before the addition of 2 mM SNAP. Results are expressed as the percentage of basal lipolysis from nontreated adipocytes (control: 35 ± 6 nmol of glycerol/105 cells/h) and are means ± S.E. of four separate experiments performed in duplicate. Statistical comparisons were made by the paired Student's t test. * p < 0.05, ** p < 0.01 versus isoproterenol; dagger  nonsignificant versus 2 mM SNAP + isoproterenol.

                              
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Table II
Effects of SNAP on cyclic AMP production stimulated by isoproterenol or forskolin in isolated adipocytes
Isolated adipocytes were incubated with increasing concentrations of SNAP in the presence of 1 µM isoproterenol or 10 µM forskolin. Numbers in parentheses refer to the values obtained in SNAP-exposed adipocytes and expressed as percentages of the control values. Results are expressed as mean ± S.E. of four separate experiments performed in duplicate. Statistical analysis was made by paired Student's t test. * p < 0.01 versus control


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Fig. 6.   SNAP does not affect forskolin-, dibutyryl cyclic AMP-, or IBMX-stimulated lipolysis. Isolated adipocytes were incubated in the presence of the indicated lipolytic agents and with or without increased SNAP concentrations. Results are expressed as the percentage of basal lipolysis from nontreated adipocytes (control: 35 ± 7 nmol of glycerol/105 cells/h) and are means ± S.E. of four separate experiments performed in duplicate.

Effects of PAPA-NONOate-- The influence of PAPA-NONOate was also studied on lipolysis stimulated by agents acting at different steps of the lipolytic cascade. As shown in Fig. 7, an important inhibitory effect of this NO donor was observed not only on lipolysis stimulated by isoproterenol but, in contrast with SNAP, on forskolin-, dibutyryl cyclic AMP-, or IBMX-stimulated responses as well. The cyclic AMP response induced by isoproterenol or forskolin in adipocytes was unaltered by PAPA-NONOate (0.5-1 mM) (Table III) indicating that the site(s) of the antilipolytic effect of PAPA-NONOate is located downstream from the receptor/G protein/adenylate cyclase step. Involvement of cyclic GMP in this antilipolytic effect of PAPA-NONOate could be ruled out since preincubation with 100 µM ODQ failed to alter the magnitude of the inhibitory effect of PAPA-NONOate on dibutyryl cyclic AMP-stimulated lipolysis (63 ± 5% versus 67 ± 5% in PAPA-NONOate and ODQ plus PAPA-NONOate treated adipocytes, respectively). To further establish the mechanism of the antilipolytic effect of PAPA-NONOate, we studied the HSL activity in adipose tissue homogenates. As shown in Table IV, PAPA-NONOate decreased by about 30% HSL activity, whatever the dose of PAPA-NONOate used, an effect which was prevented by 200 µM carboxy-PTIO, a NO scavenger.


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Fig. 7.   PAPA-NONOate decreases isoproterenol-, forskolin-, dibutyryl cyclic AMP-, and IBMX-stimulated lipolysis. Isolated adipocytes were incubated in the presence of the indicated lipolytic agents and with or without increased PAPA-NONOate concentrations. Results are expressed as the percentage of basal lipolysis from nontreated adipocytes (control: 29 ± 10 nmol of glycerol/105 cells/h) and are means ± S.E. of three separate experiments performed in duplicate. Statistical comparisons were made by the paired Student's t test. * p < 0.01, ** p < 0.001 versus the indicated lipolytic agent.

                              
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Table III
Effects of PAPA-NONOate on cyclic AMP production stimulated by isoproterenol or forskolin in isolated adipocytes
Isolated adipocytes were incubated with increasing concentrations of PAPA-NONOate in the presence of 1 µM isoproterenol or 10 µM forskolin. Numbers in parentheses refer to the values obtained in PAPA-NONOate-exposed adipocytes and expressed as percentages of the control values. Results are expressed as mean ± S.E. of three separate experiments performed in duplicate.

                              
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Table IV
Effects of PAPA-NONOate on hormone-sensitive lipase activity in adipose tissue homogenates
Adipose tissue homogenates were incubated with increasing concentrations of PAPA-NONOate with or without the NO-scavenger carboxy-PTIO (200 µM). Results are expressed as the percentages of the control (195 ± 33 nmol of oleic acid/h/mg of protein) and are means ± S.E. of five separate experiments performed in duplicate. Statistical analysis was made by paired Student's t test. * p < 0.02. 

Effects of NO Gas-- By testing various concentrations of authentic NO on isolated adipocytes, Fig. 8 shows that NO dose dependently inhibits stimulated lipolysis whatever the stimulating agent used.


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Fig. 8.   Authentic NO solutions decrease dramatically isoproterenol-, forskolin-, or dibutyryl cyclic AMP-stimulated lipolysis. Isolated adipocytes were incubated in Krebs-Ringer-Tris buffer for 1 h in the presence of the indicated lipolytic agents with increased concentrations of NO gas from a saturated buffer solution. Lipolysis is expressed as nmol of glycerol/105 cells/h. Results are means ± S.E. of three separate experiments performed in duplicate. Statistical comparisons were made by the paired Student's t test. * p < 0.01, ** p < 0.001 versus the indicated lipolytic agent.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In this study we provide the first evidence for a modulation of the lipolytic process in isolated adipocytes by NO donors and NO gas. Our results also indicate that there are at least two opposite effects of NO-related species in the control of lipolysis. Among the NO donors tested, only nitrosothiols were found to increase the rate of basal lipolysis through a mechanism apparently unrelated to cyclic GMP. Since the NO group of RS-NO has properties that enable S-nitrosylation of proteins (6, 14) one likely mechanism explaining the stimulatory effect of RS-NO on basal lipolysis could be the S-nitrosylation of SH group(s) belonging to critical protein(s) for lipolysis. Such a critical role for thiol has been previously emphasized from the observation that exposure of adipose tissue to N-ethylmaleimide (an irreversible thiol alkylating agent) results in a stimulation of basal lipolysis as well (32). As nitrosothiols are cell impermeables (27), interaction of nitrosothiols with extracellular or membraneous redox switch is likely. The particulate cGI-PDE, containing critical thiol (31), appears to be the target of SNAP as we have observed an inhibitory effect of this enzyme activity by this nitrosothiol. Our present experiment showing that the SNAP-induced stimulation of basal lipolysis was prevented by DTT (thiol-reducing agent) adds further weight to the hypothesis.

It has been shown that addition of DTT (a 1-4 dithiol) to solution of nitrosothiol accelerates the nitrosothiol decomposition into hydroxylamine (NH2OH) (14). This production of NH2OH was attributed to a nitrosation of DTT via transnitrosation followed by formation of nitroxyl anion (NO-) and disulfide (14). Thus DTT is able to modify the redox form of NO generated from nitrosothiols. From this finding and the results of the present study it can be concluded that NO+ donors increase basal lipolysis, whereas NO- generated from SNAP in the presence of DTT or NO· formed from NONOates and authentic NO· have no influence on basal lipolysis.

Whereas SNAP increases basal lipolysis, it markedly reduced isoproterenol-stimulated lipolysis. This effect seems to be linked to the decrease in cyclic AMP production observed in isoproterenol-treated adipocytes after exposure to SNAP. A direct effect of SNAP on adenylate cyclase appears unlikely since SNAP failed to decrease the forskolin-induced lipolysis. Moreover, the production of cyclic AMP was unchanged under these conditions. These results are consistent with an interaction of SNAP with the early steps of the signal transduction pathway of isoproterenol i.e. the lipolytic beta -adrenergic receptor and/or the Gs coupling protein but not with the adenylate cyclase. It is expected that S-nitrosylation or disulfide formation occurred on the beta -receptor since this receptor contains key cysteines implicated in the hormone signal transduction process (33). Additional effects of SNAP on G proteins could be also suggested as G proteins have been shown to be targets of NO in human lymphocytes (34). Moreover, DTT failed to prevent SNAP inhibition, a finding which indicates that both NO+ (in the absence of DTT) and NO- (in the presence of DTT) are able to inhibit isoproterenol-induced lipolysis. Inhibition of isoproterenol-stimulated lipolysis by SNAP could also be mediated by NO· as the homolytic cleavage of the S-N bound of SNAP generates NO· (14). This possibility seems to be ruled out, however, since NO· donors like NONOates failed to alter isoproterenol-stimulated cyclic AMP production in adipocytes.

The mechanism(s) whereby NO· (issue from authentic NO or NONOates) inhibits lipolysis clearly appears different from that underlying the antilipolytic effect of nitrosothiols, although both are cyclic GMP-independent. As the matter of fact, NO· elicited a marked antilipolytic effect regardless of the stimulatory agent used and NO· failed to reduce the cyclic AMP responses to isoproterenol or forskolin. Moreover, the marked inhibition induced by NO· on dibutyryl cyclic AMP- or IBMX-stimulated lipolysis excludes the possibility that an activation of phosphodiesterases could be involved in the inhibitory mechanism of NO·. A direct effect of NO· on HSL activity can be suggested as PAPA-NONOate slightly decreased the HSL activity. It appears thus that NO· probably interferes at the level of the HSL but also on the cyclic AMP-dependent activation of cyclic AMP-dependent protein kinase and/or on cyclic AMP-dependent protein kinase-dependent HSL phosphorylation by a mechanism that remains to be determined.

Under physiological conditions, NO can be interconverted among different redox forms with distinctive chemistries (4). The present results obtained with different NO donors suggest that the intracellular or extracellular redox states are important factors in determining the type of response of the lipolytic process to NO. An increase in basal lipolysis can be observed with nitrosothiols. Nitrosothiols do occur naturally in human plasma mainly as the nitrosothiol of human serum albumin (5). S-nitrosoglutathione has been identified in normal airways (35) and in neutrophils (36). Concerning the stimulated lipolysis, NO-related species inhibit the beta -adrenergic-stimulated lipolysis. However, the inhibitory mechanisms are depending on the NO form. Such inhibition of catecholamine-induced lipolysis could have important physiological metabolic consequences as lipolysis is mainly controlled by the sympathetic nervous system activity and the plasma insulin levels. NO produced by white adipocytes may function as an autocrine regulator of catecholamine-stimulated lipolysis.

In summary, we have identified a potentially important modulation of lipolysis by NO. Our findings indicate that NO activates or inhibits lipolysis through cyclic GMP-independent mechanisms that are tighly linked to the redox state of NO.

    ACKNOWLEDGEMENTS

We thank Dr. J. L. Boucher, URA 400 CNRS, Université René Descartes for useful discussions and kindly providing NO gas.

    FOOTNOTES

* This work was supported by grants from the INSERM, the University René Descartes (Paris-V), and the Ligue Nationale contre le Cancer (Délégation Départementale des Yvelines).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.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, 45 rue des Saints Pères, 75270 Paris Cedex 06, France. Tel.: 33-1-42-86-22-01; Fax: 33-1-42-86-04-02.

1 The abbreviations used are: NO, nitric oxide; NOS, nitric oxide synthase; SNAP, S-nitroso-N-acetyl-DL-penicillamine; RS-NO, S-nitrosothiol; GS-NO, S-nitrosoglutathione; DTT, dithiothreitol; ODQ, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; PAPA-NONOate, 1-propamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazine); ISO, (-)-isoproterenol; carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; IBMX, 3-isobutyl-1-methylxanthine; PDE, phosphodiesterase; cGI-PDE, cyclic GMP-inhibited low Km cyclic AMP PDE; HSL, hormone-sensitive lipase; KRT, Krebs-Ringer-Tris buffer.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Pharmacol. Rev. 43, 109-142[Medline] [Order article via Infotrieve]
  2. Lowenstein, C. J., Dinerman, J. L., and Snyder, S. H. (1994) Ann. Intern. Med. 120, 227-237[Abstract/Free Full Text]
  3. Ignarro, L. J. (1990) Pharmacol. Toxicol. 67, 1-7[Medline] [Order article via Infotrieve]
  4. Stamler, J. S., Singel, D. J., and Loscalzo, J. (1992) Science 258, 1898-1902[Medline] [Order article via Infotrieve]
  5. Stamler, J. S., Simon, D. I., Osborne, J. A., Mullins, M. E., Jaraki, O., Michel, T., Singel, D. J., and Loscalzo, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 444-448[Abstract]
  6. Stamler, J. S. (1995) Curr. Top. Microbiol. Immunol. 196, 19-35[Medline] [Order article via Infotrieve]
  7. Förstermann, U., Gath, I., Schwarz, P., Closs, E. I., and Kleinert, H. (1995) Biochem. Pharmacol. 50, 1321-1332[CrossRef][Medline] [Order article via Infotrieve]
  8. Ribière, C., Jaubert, A. M., Gaudiot, N., Sabourault, D., Marcus, M. L., Boucher, J. L., Denis-Henriot, D., and Giudicelli, Y. (1996) Biochem. Biophys. Res. Commun. 222, 706-712[CrossRef][Medline] [Order article via Infotrieve]
  9. Hotamisligil, G. S., Shargill, N. S., and Spiegelman, B. M. (1993) Science 259, 87-91[Medline] [Order article via Infotrieve]
  10. Kawakami, M., Murase, T., Ogawa, H., Ishibashi, S., Mori, N., Takaku, F., and Shibata, S. (1987) J. Biochem. 101, 331-338[Abstract]
  11. Grunfeld, C., Gulli, R., Moser, A. H., Gavin, L. A., and Feingold, K. R. (1989) J. Lipid Res. 30, 579-585[Abstract]
  12. Green, A., Dobias, S. B., Walters, D. J. A., and Brasier, A. R. (1994) Endocrinology 134, 2581-2588[Abstract]
  13. Hauner, H., Petruschke, Th, Russ, M., Röhrig, K., and Eckel, J. (1995) Diabetologia 38, 764-771[CrossRef][Medline] [Order article via Infotrieve]
  14. Arnelle, D. R., and Stamler, J. S. (1995) Arch. Biochem. Biophys. 318, 279-285[CrossRef][Medline] [Order article via Infotrieve]
  15. Feelisch, M., and Stamler, J. S. (1996) in Methods in Nitric Oxide Research (Feelisch, M., and Stamler, J. S., eds), pp. 71-115, John Wiley & Sons, Chichester, West Sussex, England
  16. Giudicelli, Y., Provin, D., and Nordmann, R. (1975) Biochimie (Paris) 58, 979-981
  17. Rodbell, M. (1964) J. Biol. Chem. 239, 375-380[Free Full Text]
  18. Wieland, O. H. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H., ed), 3rd Ed., Vol. VI, pp. 504-510, Verlag Chemie, Weinheim, Germany
  19. Khoo, J. C., and Steinberg, D. (1975) Methods Enzymol. 35, 181-189[Medline] [Order article via Infotrieve]
  20. Belfrage, P., and Vaughan, M. (1969) J. Lipid Res. 10, 341-344[Abstract/Free Full Text]
  21. Kono, T. (1988) Methods Enzymol. 159, 745-751[Medline] [Order article via Infotrieve]
  22. Feelisch, M., Kubitzek, D., and Werringloer, J. (1996) in Methods in Nitric Oxide Research (Feelisch, M., and Stamler, J. S., eds), pp. 455-478, John Wiley & Sons, Chichester, West Sussex, England
  23. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  24. Hirsh, J., and Gallian, A. (1968) J. Lipid Res. 9, 110-119[Abstract/Free Full Text]
  25. Akaike, T., Yoshida, M., Miyamoto, Y., Sato, K., Kohno, M., Sasamoto, K., Miyazaki, K., and Maeda, H. (1993) Biochemistry 32, 827-832[Medline] [Order article via Infotrieve]
  26. Garthwaite, J., Southam, E., Boulton, C. L., Nielsen, E. B., Schmidt, K., and Mayer, B. (1995) Mol. Pharmacol. 48, 184-188[Abstract]
  27. Campbell, D. L., Stamler, J. S., and Strauss, H. C. (1996) J. Gen. Physiol. 108, 277-293[Abstract]
  28. Loten, E. G., and Sneyd, J. G. T. (1970) Biochem. J. 120, 187-193[Medline] [Order article via Infotrieve]
  29. Manganiello, V. C., and Vaughan, M. (1973) J. Biol. Chem. 248, 7164-7170[Abstract/Free Full Text]
  30. Elks, M. L., Manganiello, V. C., and Vaughan, M. (1983) J. Biol. Chem. 258, 8582-8587[Abstract/Free Full Text]
  31. Degerman, E., Belfrage, P., Newman, A. H., Rice, K. C., and Manganiello, V. C. (1987) J. Biol. Chem. 262, 5797-5807[Abstract/Free Full Text]
  32. Giudicelli, Y., Provins, D., and Nordmann, R. (1975) Biochem. Pharmacol. 24, 1029-1033[Medline] [Order article via Infotrieve]
  33. Savarese, T. M., and Fraser, C. M. (1992) Biochem. J. 283, 1-19[Medline] [Order article via Infotrieve]
  34. Lander, H. M., Sehajpal, P. K., and Novogrodsky, A. (1993) J. Immunol. 151, 7182-7187[Abstract/Free Full Text]
  35. Gaston, B., Reilly, J., Drazen, J. M., Fackler, J., Ramdev, P., Arnelle, D., Mullins, M., Sugarbaker, D. J., Chee, C., Singel, D. J., Loscalzo, J., and Stamler, J. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10957-10961[Abstract]
  36. Clancy, R. M., Yegudin, J., Levartovsky, D., Piziak-Leszcynska, J., and Abramson, S. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3680-3684[Abstract]


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