From the Department of Biochemistry, INSERM CJF 94--02, Faculté de Médecine de Paris-Ouest, Universite René
Descartes, Paris, France
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INTRODUCTION |
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
is expressed and
secreted by adipose tissue, which is also an important target of this
cytokine (9). Indeed tumor necrosis factor
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
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
-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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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; 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.
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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.
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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.
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Effects of NO Donors and Authentic NO on Stimulated
Lipolysis--
Lipolysis was stimulated using different agents acting:
(i) at the
-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
-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; 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.
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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.
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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.
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DISCUSSION |
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
-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
-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
-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.
We thank Dr. J. L. Boucher, URA 400 CNRS, Université René Descartes for useful discussions and
kindly providing NO gas.