Leptin-induced nitric oxide production in white adipocytes is mediated through PKA and MAP kinase activation

Nadia Mehebik, Anne-Marie Jaubert, Dominique Sabourault, Yves Giudicelli, and Catherine Ribière

Department of Biochemistry and Molecular Biology (UPRES EA-2493), Faculty of Médecine Paris-Ile de France-Ouest, University of Versailles Saint-Quentin en Yvelines, Paris, France

Submitted 6 July 2004 ; accepted in final form 8 March 2005


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Leptin injection increases plasma levels of nitrites and/or nitrates, an index of nitric oxide (NO) production. Because plasma levels of NO are correlated with fat mass and because adipose tissue is the main source of leptin, it seems that adipose tissue plays a major role in NO release induced by leptin. Adipocytes express both leptin receptors and nitric oxide synthase (NOS; including the endothelial isoform, NOS III, and the inducible isoform, NOS II). In this study, we have demonstrated that physiological concentrations of leptin stimulate NOS activity in adipocytes. This effect of leptin is abolished by 1) AG490, an inhibitor of Janus tyrosine kinase 2/signal transducer and activator of transcription 3; 2) U0126, an inhibitor of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (p42/p44 MAPK); and 3) N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) or Rp diastereomer of adenosine 3',5'-cyclic phosphorothioate, two inhibitors of protein kinase A, but not by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Immunoblotting studies have shown that leptin fails to activate Akt but increases p42/p44 MAPK phosphorylation, an effect that is prevented by U0126 but not by H-89. Furthermore, leptin induces NOS III phosphorylation at Ser1179 and Thr497, but not when adipocytes are pretreated with H-89 or U0126. Finally, stimulation of adipocyte NOS activity by leptin is either unaltered when protein phosphatase 2A is inhibited by 1 nM okadaic acid or completely abolished when protein phosphatase 1 (PP1) activity is inhibited by 3 nM tautomycin, which supports a crucial role for PP1 in mediating this effect of leptin. On the whole, these experiments demonstrate that NOS activity is a novel target for leptin in adipocytes and that the leptin-induced NOS activity is at least in part the result of NOS III phosphorylations via both protein kinase A and p42/p44 MAPK activation. More generally, this study also leads to the hypothesis of NO as a potentially important factor for leptin signaling in adipocytes.

protein kinase A; p42/p44 mitogen-activated protein kinase


LEPTIN, THE PRODUCT OF THE ob gene essentially secreted by white adipocytes, appears to play a major role in the control of body fat stores through coordinated regulation of feeding behavior, body energy balance, and neuroendocrine responses (19). Leptin is structurally related to cytokines and binds to specific receptors that belong to the class I cytokine receptor superfamily (47). Several alternative spliced isoforms of leptin receptors (Ob-R) have been described and cloned (48). The major leptin signaling Ob-R is the long isoform Ob-Rb. Analogously to other cytokine receptor transduction systems, leptin stimulation results in activation of the intracellular Janus tyrosine kinases (JAKs) such as JAK2 and leads to phosphorylation of cytoplasmic target proteins, including the signal transducer and activator of transcription (STAT), the ras/mitogen-activated protein kinase (MAPK), and the phosphatidylinositol 3-kinase (PI3-kinase) pathways (28). Besides the importance of the leptin metabolic effects initiated primarily in the hypothalamus, this hormone elicits various peripheral actions (22). The existence of functional leptin receptors in white adipose tissue raises the possibility of leptin autocrine-paracrine effects on this tissue. In fact, leptin is directly involved in the regulation of both adipose tissue metabolism [inhibition of lipogenesis (2, 50) and stimulation of lipolysis (20, 21, 50)] and adipogenesis (34). It was further suggested that nitric oxide (NO) is a potential regulator of leptin-induced lipolysis (23). Indeed, leptin administration increases both lipolysis and the plasma levels of NO3-NO2 (an index of NO production), two effects that are reduced under conditions of nitric oxide synthase (NOS) inhibition (23). Because plasma leptin and NO levels are directly related to fat mass, it was suggested that adipocytes play a major role in NO release induced by leptin (35).

We have previously shown that rat adipose tissue expresses two isoforms of NOS, the endothelial isoform (NOS III) and the inducible isoform (NOS II), and that NO plays an important role in lipolysis regulation at least in vitro (24, 25, 41).

NOS III activity is regulated by reversible phosphorylation through multiple protein kinases [e.g., AMP-activated protein kinase (AMPK), Akt, PKA, PKC] and protein phosphatases acting on Ser1179 and Thr497 (18, 37, 44). In various tissues, these protein kinases were found to be involved in leptin signaling (46). In white adipocytes that express two leptin receptor isoforms, Ob-Ra and Ob-Rb (34), it thus seems likely that leptin also influences NOS III activity through phosphorylation-dependent mechanisms. The aim of the present study was to test this hypothesis and to establish precisely the mechanisms involved.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Murine leptin was purchased from PeproTech (London, UK), and L-[2,3,4,5-3H]arginine (58 Ci/mmol) and the ECL detection kit were products of Amersham Biosciences (Little Chalfont, UK). AG 50W-X8, cation exchange resin, Bradford protein dye reagent, and electrophoretic chemicals were purchased from Bio-Rad Laboratories (Hercules, CA) and tautomycin was obtained from Biomol International (Plymouth Meeting, PA). Anti-active MAP kinase (V8031) and anti-phospho-Akt (G7441) antibodies were obtained from Promega (Madison, WI). U0126 and anti-phospho-Ser1179 NOS III antibody were purchased from Cell Signaling Technology (Beverly, MA), monoclonal anti-phospho-NOS III Thr495 antibody was obtained from Upstate Cell Signaling Solutions (Charlottesville, VA), and polyclonal anti-NOS III antibody and ERK (pan-ERK) antibody were purchased from BD Transduction Laboratories (San Diego, CA). Other reagents were obtained from Sigma (St. Louis, MO).

Animals. Male Sprague-Dawley rats (180–200 g) were obtained from Centre d'Elevage de Rats Janvier (Le Genest St. Isle, France) at 8 wk of age and maintained at constant room temperature (24°C) on a 12:12-h light-dark cycle. Fed rats were killed by decapitation, and white adipose tissue from epididymal fat deposits were carefully removed and rapidly used for adipocyte preparation. All experimental protocols were approved by the University Animal Use and Care Committee.

Adipocytes incubation and NOS activity. Isolated epididymal adipocytes were prepared as previously described (41). NOS activity was measured after L-[3H]arginine conversion into L-[3H]citrulline by intact adipocytes (25). Briefly, adipocytes (3–5 x 105 cells/ml) were incubated at 37°C in Krebs-Ringer HCO3 (KRB; pH 7.4) containing 2% (wt/vol) BSA, 5 mM glucose, 1.5 µCi/ml L-[3H]arginine, and 50 mM valine (to inhibit arginase) in the absence or presence of various concentrations of leptin and/or the effectors to be tested. Each incubation was performed without and with a specific NOS inhibitor, diphenyliodonium (DPI). After 30 min, incubations were stopped by adding 250 µl of ethanol followed by 5 ml of 1:1 (vol/vol) H2O/Dowex 50W-X8 (Na+ form) resin and then were left to settle for 10 min at 4°C. One milliliter of supernatant was then removed and added to liquid scintillation cocktail for counting. Values obtained in the presence of DPI were subtracted from each sample. DMSO, which was used as vehicle for the tested inhibitors, was added to controls, and separate experiments revealed no effect of this compound on the adipocyte NOS activity.

Cell lysates. Adipocytes (3–5 x 105 cells/ml) were incubated during 20 min at 37°C in KRB (pH 7.4) containing 5 mM glucose in the absence or presence of the effectors to be tested. Next, adipocytes were harvested and disrupted in buffer containing 20 mM Tris, pH 7.5, 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1% Nonidet P-40, and protease inhibitors (30). Cell lysates were solubilized by continuous stirring for 30 min at 4°C and centrifuged for 10 min at 14,000 g. Supernatants were used for protein determination according to the Bradford assay method (8) and, after Laemmli buffer addition, for electrophoresis and immunoblotting studies.

Western blot analysis. Cell lysates (10–20 µg of protein/lane) were subjected to SDS-PAGE and then blotted onto polyvinylidene difluoride membranes. The blots were incubated with the primary antibody at 4°C overnight and incubated with the secondary antibody linked to peroxidase. Immunoreactive proteins were visualized on X-ray film using ECL reagents. Immunoblotting was performed using specific anti-active MAP kinase (phospho-Thr184 and phospho-Tyr185), anti-ERK (pan-ERK), anti-phospho-Akt (Ser473), anti-phospho-NOS III (Ser1179), anti-phospho-NOS III (Thr497), and anti-NOS III antibodies. All experiments were performed at least in triplicate.

Statistical analysis. Data are presented as means ± SE. Statistical analyses were performed using an unpaired Student's t-test.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Leptin increases NOS activity. Adipocyte exposure to leptin resulted in a dose-dependent increase in NOS activity, which reaches its maximum at the concentration of 1 ng/ml (Fig. 1A), in contrast to a previous study in which supraphysiological leptin concentrations were required (35). The signal transduction mechanisms involved in this effect of leptin were next studied using 10 ng/ml, a leptin concentration frequently observed in animal and human plasma. Because the leptin signal is thought to be transmitted mainly by the JAK2/STAT3 pathway, we tested the effect of a selective JAK2/STAT3 inhibitor, AG490 (33), on leptin-stimulated NOS activity. As shown in Fig. 1B, AG490 alone did not alter basal NOS activity but prevented the leptin effect.



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Fig. 1. Leptin increases nitric oxide (NO) synthase (NOS) activity in rat adipocytes, an effect prevented by an inhibitor of the Janus tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. A: isolated adipocytes were incubated with increasing concentrations of leptin (L; 0.1–1,000 ng/ml) for 30 min. B: adipocytes were pretreated 15 min without or with AG490 (10–5 M), an inhibitor of JAK2, and then exposed to vehicle or leptin (10 ng/ml) for another 30 min. NOS activity was measured by the conversion of L-[3H]arginine into L-[3H]citrulline as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of independent experiments performed in duplicate with four separate adipocyte preparations and are expressed as %NOS activity in control adipocytes (C). *P < 0.02. **P < 0.01 vs. control.

 
Leptin stimulation of NOS is independent of PI3-kinase/Akt activation. Recent studies have demonstrated that in response to various hormones (18), NOS III becomes activated after phosphorylation on Ser1179 by Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3-kinase). Thus we tested the influence of a specific PI3-kinase inhibitor, wortmannin, on adipocyte NOS activity. As shown in Fig. 2A, wortmannin fails to influence both basal and leptin-stimulated NOS activity. Akt activation after adipocyte exposure to various leptin concentrations was next examined using Western blot analysis. Data in Fig. 2B clearly show the failure of leptin to activate Akt regardless of the concentrations used, which is in contrast to the Akt activation induced by 1 nM insulin used as positive control. These results indicate that the PI3-kinase/Akt pathway is not involved in the mechanism whereby leptin stimulates NOS activity in adipocytes.



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Fig. 2. Phosphatidylinositol 3-kinase (PI3-kinase) and Akt activation are not involved in leptin-induced NOS activity. A: adipocytes were pretreated with vehicle alone or PI3-kinase inhibitor, 10–6 M wortmannin (W), for 15 min, and then exposed to vehicle or leptin (10 ng/ml) for another 30 min. NOS activity was assessed as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of independent experiments performed in duplicate with four separate adipocyte preparations and are expressed as %NOS activity in control adipocytes (C). *P < 0.05 vs. control. B: adipocytes were incubated with leptin (10 or 100 ng/ml) or insulin (I; 1 nM), used as a positive control, for 10 min. Cell lysates, prepared as described in EXPERIMENTAL PROCEDURES, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed using an antibody specific for the phosphorylated form of Akt (Ser473). Results are representative of three separate experiments.

 
Leptin stimulation of NOS involves PKA activation. The above-described findings prompted us to consider alternative mechanisms such as PKA activation. Thus we pretreated adipocytes with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a PKA inhibitor, before leptin incubation. Interestingly, H-89, which had no effect on NOS activity per se, completely abolished leptin-stimulated NOS activity (Fig. 3A). Pretreatment of adipocytes with Rp diastereomer of adenosine 3',5'-cyclic phosphorothioate (Rp-cAMPS), which blocks the PKA activation (43), also prevented the leptin-induced NOS activity (Fig. 3B). Because leptin was shown to increase cAMP in adipocytes (29), we further examined the role of PKA activation in NOS activity using a cell-permeable analog of cAMP (dibutyryl adenosine 3',5'-cyclic monophosphate, DBcAMP). Like leptin, this compound significantly stimulated NOS activity in adipocytes, an effect that was prevented by H-89 (Fig. 4A). Moreover, simultaneous addition of leptin and DBcAMP did not modify the stimulation of NOS activity produced by each of these compounds added alone (Fig. 4B). Therefore, these results indicate an important role for PKA in NOS activation by leptin in adipocytes.



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Fig. 3. Protein kinase A inhibitors block leptin-induced NOS activity. Adipocytes were pretreated with vehicle alone or with PKA inhibitors, 10–5 M N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; A) and 10–4 M Rp diastereomer of adenosine 3',5'-cyclic phosphorothioate (Rp-cAMPS; B), for 15 min, and then exposed to vehicle or leptin (10 ng/ml) for another 30 min. NOS activity was measured as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of independent experiments performed in duplicate with four separate adipocyte preparations and are expressed as %NOS activity in control adipocytes (C). **P < 0.01 vs. control.

 


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Fig. 4. Dibutyryl cAMP (DBcAMP) stimulates NOS activity in adipocytes. A: adipocytes were incubated without or with H-89 (10–5 M), an inhibitor of PKA, for 15 min and then exposed to vehicle or DBcAMP (10–3 M) for another 30 min. B: adipocytes were exposed to leptin (10 ng/ml), DBcAMP (10–3 M) alone, or leptin plus DBcAMP. NOS activity was measured by the conversion of L-[3H]arginine into L-[3H]citrulline as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of independent experiments performed in duplicate with five separate adipocyte preparations and are expressed as %NOS activity in control adipocytes (C). *P < 0.05. **P < 0.01 vs. control. ns, not significant.

 
Leptin stimulation of NOS involves MAP kinase activation. We next tested the eventual role of MAPK in leptin-induced NOS activation. As previously reported by our laboratory (34), in rat adipocytes, leptin activated p42/p44 MAPK without regard to the concentration tested (Fig. 5A). We also found that MAPK activation by leptin is blocked by U0126, a selective inhibitor of MEK, the immediate upstream activator of p42/p44 MAPK, but not by H-89 (Fig. 5B). Furthermore, U0126, which failed to alter basal NOS activity, completely prevented leptin-induced NOS activity (Fig. 5C). These observations support that NOS is directly or indirectly a target for leptin via activation of the MAPK pathway.



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Fig. 5. Leptin activation of NOS requires mitogen-activated protein kinase (MAPK) stimulation. A: adipocytes were incubated with leptin (1–100 ng/ml) for 20 min. B: adipocytes were pretreated with vehicle alone or with a MEK inhibitor, U0126 (10–5 M) or H-89 (10–5 M), for 15 min, and then exposed to vehicle or leptin (10 ng/ml) for another 20 min. Cell lysates, prepared as described in EXPERIMENTAL PROCEDURES, were separated on SDS-12% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed using an antibody specific for the phosphorylated form of p42/p44 MAPK or with anti-total p42/p44 MAPK antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SE of independent experiments performed in duplicate with three separate adipocyte preparations. *P < 0.05 vs. control. C: adipocytes were pretreated with vehicle alone or with U0126 (10–5 M) for 15 min, and then NOS activity was assessed as described in EXPERIMENTAL PROCEDURES in adipocytes exposed to vehicle or to leptin (10 ng/ml) for 30 min. Results are means ± SE of independent experiments performed in duplicate with four separate adipocyte preparations and are expressed as %NOS activity in control adipocytes (C). *P < 0.05 vs. control.

 
Leptin phosphorylates NOS III on Ser1179 and Thr497. Because NOS III (but not NOS II) is a target for protein kinases such as PKA (9), it was important to determine whether leptin stimulation of NOS activity in adipocytes implies phosphorylation of NOS III at Ser1179, which is crucial for the regulation of this enzyme catalytic activity (17). Adipocytes were incubated with leptin or with insulin for 20 min, which was used as a positive control, and phosphorylation was detected by performing Western blot analysis using a specific anti-phospho-Ser1179 NOS III antibody. As shown in Fig. 6A, adipocyte exposure to leptin or to insulin resulted in a clear increase in NOS III phosphorylation at Ser1179. Furthermore, DBcAMP, like leptin, also increased this NOS III phosphorylation (Fig. 6C). Because, as we have shown, PKA and MAPK are implicated in the leptin-induced NOS activity, we tested the two protein kinase inhibitors, H-89 and U0126, on the phosphorylation of Ser1179. Both leptin- and DBcAMP-induced phosphorylation at Ser1179 were prevented by H-89 (Fig. 6, B and C). In contrast, U0126 prevented NOS III phosphorylation at Ser1179 induced by leptin (Fig. 6B), but not that induced by DBcAMP (Fig. 6C). Wortmannin was unable to modify DBcAMP NOS III phosphorylation (Fig. 6C), which is quite different from the situation reported in endothelial cells (5).



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Fig. 6. Leptin or DBcAMP induces phosphorylation of NOS III at Ser1179. A: adipocytes were incubated with leptin (10 ng/ml) or insulin (1 nM), used as a positive control, for 20 min. B: adipocytes were pretreated 15 min without or with H-89 (10–5 M), an inhibitor of PKA, or with U0126 (10–5 M), a MEK inhibitor, and then exposed to vehicle or leptin (10 ng/ml) for another 20 min. C: adipocytes were pretreated for 15 min without or with H-89 (10–5 M), U0126 (10–5 M), or wortmannin (10–6 M) and then incubated with vehicle or DBcAMP (10–3 M) for another 20 min. Cell lysates, prepared as described in EXPERIMENTAL PROCEDURES, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed using an antibody specific for the phosphorylated form of NOS III at Ser1179 and polyclonal anti-NOS III antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SE of independent experiments performed in duplicate with three separate adipocyte preparations. *P < 0.05. **P < 0.01 vs. control.

 
Because PKA could also phosphorylate NOS III on Thr497 (9), we decided to determine whether NOS III phosphorylation at this site is also regulated by leptin or DBcAMP. Leptin increased NOS III phosphorylation on Thr497 (Fig. 7A) and H-89, or U0126 prevented this leptin effect (Fig. 7A). DBcAMP also induced Thr497 phosphorylation, but only H-89 blocked this DBcAMP effect (Fig. 7B). The finding that NOS III phosphorylation on Thr497 is increased by DBcAMP in adipocytes also is in contrast to the endothelial cell model, wherein cAMP was shown to cause coordinated Thr497 dephosphorylation and enhanced Ser1179 phosphorylation of NOS III (5, 37). Nevertheless, these results indicate that PKA is able to phosphorylate adipocyte NOS III not only on Ser1179 but also on Thr497 NOS III, which is consistent with previous experiments performed with purified NOS III (9).



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Fig. 7. Leptin or DBcAMP induces phosphorylation of NOS III at Thr497. A: adipocytes were pretreated for 15 min without or with H-89 (10–5 M), an inhibitor of PKA, or with U0126 (10–5 M), a MEK inhibitor, and then exposed to vehicle or to leptin (10 ng/ml) for another 20 min. B: adipocytes were pretreated for 15 min without or with H-89 (10–5 M) or U0126 (10–5 M) and then exposed to vehicle or DBcAMP (10–3 M) for another 20 min. Cell lysates, prepared as described in EXPERIMENTAL PROCEDURES, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed using an antibody specific for the phosphorylated form of NOS III at Thr497 and an anti-polyclonal NOS III antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SE of independent experiments performed in duplicate with three separate adipocyte preparations. *P < 0.05. **P < 0.01 vs. control.

 
Protein phosphatase 1 modulates leptin-induced Thr497 phosphorylation on NOS III. Previous reports have demonstrated that besides Ser1179 phosphorylation, regulation of NOS III activity implicated two Ser/Thr protein phosphatases: protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) (37). PP1 is responsible for Thr497 dephosphorylation, which results in NOS III activation, while PP2A seems to catalyze Ser1179 dephosphorylation, leading to enzyme inactivation. To date, leptin has not been reported to regulate PP1 or PP2A activities. Nevertheless, we decided to investigate the influence of okadaic acid (OA; a PP1 and/or PP2A inhibitor) on leptin-stimulated NO production. At 1 nM, a concentration reported to specifically inhibit PP2A (39, 42), OA did not modify leptin-stimulated NO production (Fig. 8A). In contrast, at 1 µM, a concentration inhibiting both PP2A and PP1, OA stimulated basal NOS activity as previously observed in adipocytes (42), but it also reduced NOS activity induced by leptin to a level significantly lower than that of the control (Fig. 8B). These results led us to further investigate the role of Ser1179 and Thr497 phosphorylation in leptin-mediated NOS III activation. Treatment of adipocytes with 1 µM OA alone increased Ser1179 phosphorylation approximately threefold and Thr497 twofold (Fig. 9, A and B). Leptin did not modify the Ser1179 and Thr497 phosphorylation induced by 1 µM OA. Thus these results cannot explain the reduced NOS III activity observed in the presence of both leptin and 1 µM OA. Because 1 µM OA was also reported to increase Akt phosphorylation through PP2A inhibition (40), one possible explanation for the reduced basal NOS III activity observed with 1 µM OA plus leptin could be a negative interaction between the pathways used by leptin and OA to stimulate NOS III activity. Regardless of this hypothesis, and to gain further information regarding the role of PP-1 in the leptin signaling pathway, we next tested another PP-1 inhibitor, tautomycin, at 3 nM, a concentration inhibiting only PP1 (39). Tautomycin caused an increase in Thr497 phosphorylation (Fig. 9B) and enhanced the leptin-induced phosphorylation at Thr497. Tautomycin alone also increased Ser1179 phosphorylation by a mechanism not related to PP2A inhibition, while in the presence of leptin, it strongly reduced the Ser1179 phosphorylation of NOS III (Fig. 9A). Finally, 3 nM tautomycin prevented the NOS activity induced by leptin (Fig. 8C). On the whole, these data suggest a crucial role played by PP1 in leptin-induced NOS III activation.



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Fig. 8. Protein phosphatase 1 (PP1) inhibition prevents leptin-induced NOS activity. A: adipocytes were pretreated for 15 min without or with 1 nM okadaic acid (OA), a concentration specifically inhibiting protein phosphatase 2A (PP2A), and then exposed to vehicle or to leptin (10 ng/ml) for another 30 min. B: adipocytes were pretreated for 15 min without or with 1 µM OA, a concentration inhibiting both PP1 and PP2A, and then were exposed to vehicle or to leptin (10 ng/ml) for another 30 min. C: adipocytes were pretreated for 15 min without or with 3 nM tautomycin (TAU), a concentration specifically inhibiting PP1, and then incubated with vehicle or leptin (10 ng/ml) for another 30 min. NOS activity was measured by converting L-[3H]arginine into L-[3H]citrulline as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of independent experiments performed in duplicate with four separate adipocyte preparations and are expressed as %activity in control adipocytes (C). **P < 0.01 vs. control.

 


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Fig. 9. Inhibition of PP1 alone alters NOS phosphorylations induced by leptin. Adipocytes were pretreated for 15 min without or with 1 µM OA or 3 nM TAU and then exposed to vehicle or to leptin (10 ng/ml) for another 20 min. Cell lysates, prepared as described in EXPERIMENTAL PROCEDURES, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed using an antibody specific for the phosphorylated form of NOS III at Ser1179 (A) or at Thr497 (B) and using a polyclonal anti-NOS III antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SE of independent experiments performed in duplicate with three separate adipocyte preparations. *P < 0.05. **P < 0.01 vs. control. §P < 0.05 vs. TAU + leptin.

 

    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study has demonstrated that in white adipocytes, leptin-induced NOS activation is mediated by JAK2, PKA, and MAP kinases. Because only the long isoform Ob-Rb mediates leptin-dependent tyrosine phosphorylation of JAK2 (1, 26, 31), it appears that this isoform plays a major role in the leptin-induced NOS activity. The major finding of our present study is that leptin induces phosphorylation of NOS III on Ser1179 and Thr497. Phosphorylation of NOS III at key regulatory sites plays an important regulatory role, especially in the response of this enzyme activity to various physiological stimuli (17). Ser1179 is the target of multiple protein kinases, including Akt, AMPK (13), PKA, protein kinase G (9), and CaMII protein kinase (18). Mechanistically, NOS III phosphorylation on Ser1179 increases NO release by enhancing the rate of electron flux through the reductase domain of NOS III and by improving the Ca2+ sensitivity of the enzyme (36). In the present study, we have observed that leptin did not induce Akt phosphorylation. Furthermore, leptin-induced NOS activation was not prevented under conditions inhibiting PI3-kinase, an upstream kinase inducing Akt activation. This finding is in contrast to the findings of previous studies suggesting that the mechanism whereby leptin induces endothelial cell NOS III activity implies activation of a PI3-kinase-independent Akt-NOS III phosphorylation pathway (49). Nevertheless, Vecchione et al. (49) did not provide direct evidence that Akt was indeed the protein kinase directly responsible for NOS III phosphorylation at Ser1179 in endothelial cells. Moreover, we cannot exclude that signaling pathways used by leptin to stimulate NOS activity may differ according to cell phenotype. With regard to adipocytes that express NOS II and NOS III (41), our results show that PKA plays a pivotal role in NOS activation in response to DBcAMP or leptin, because H-89 prevented both NOS activation and NOS III phosphorylation at Ser1179 and Thr497 induced by these compounds. Because NOS III, but neither NOS I nor NOS II, is rapidly activated after phosphorylation on both Ser and Thr by the catalytic subunit of PKA (9), it appears more likely that the leptin-induced NOS activity is related primarily to NOS III activation. In bovine aortic endothelial cells, shear stress was shown to stimulate NO production through Akt-dependent mechanisms (27), but Akt was not responsible for NOS III phosphorylation at Ser1179 (5). Indeed, PKA was found to be responsible for NOS phosphorylation at Ser1179 and Ser635 and subsequently for the increased NOS III activity in response to shear stress (5, 6). Therefore, if Akt is generally recognized as the key regulator of NO production through NOS III phosphorylation, our present results, as well as results reported elsewhere (5, 6), indicate that PKA is a new alternative partner in the phosphorylation-dependent NOS III activation in response to various stimuli such as shear stress or leptin. However, this does not exclude that PKA requires other signal transduction pathways, such as Akt or MAPK, for NOS III activation.

In adipose tissue, leptin was shown to increase lipolysis (20, 21, 23, 29). This prevention of this lipolytic effect by H-89 seems to be mediated by PKA (29). Therefore, it appears that leptin, through PKA activation, can increase both NO production and lipolysis. Involvement of another signaling partner such as AMPK seems unlikely, because activation of AMPK with 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (0.5–5 mM) was 1) unable to stimulate NO production in adipocytes (results not shown) and 2) found to decrease lipolysis (45). In a previous study (24), we showed that NO increased basal lipolytic activity. If leptin, through PKA activation, is able to increase lipolysis, NO production induced by leptin can also contribute to this lipolytic response by protecting the cAMP lipolytic process, and especially PKA (25), against reactive oxygen species whose generation is increased by leptin (7, 51). This hypothesis is strengthened by the finding that the stimulatory effect of leptin injection on lipolysis is 1) correlated with serum leptin concentration and 2) decreased under conditions inducing NOS inhibition (23). It thus appears that leptin, via PKA, can modulate various converging metabolic processes.

In addition to PKA, we have confirmed herein that leptin activates p42/p44 MAPK in adipocytes (34) and have shown that inhibition of these kinases blocks leptin-induced NOS activation. p42/p44 MAPK is essential for NOS III activation by several agonists, including estrogen, insulin, shear stress, H2O2, and high-density lipoprotein (10, 12, 15, 38, 42). However, the mechanism whereby MAPK is involved in NOS III activation remains unclear, but it does not appear to be related in any way to Ser1179 phosphorylation of NOS III after various stimuli (6, 10, 38). In the present study, NOS III phosphorylation induced by leptin (but not by DBcAMP) was prevented by MAPK inhibition, indicating that in addition to PKA, MAPK activation also is required for leptin-induced NOS III activation and phosphorylation. The latter finding indicates that a physiological stimulus such as leptin is not necessarily identical to a bolus addition of DBcAMP. Because in vitro p42/p44 MAPK activation induces NOS III phosphorylation on undefined sites other than Ser1179 and also decreases NOS activity (4), the role of MAPK in the leptin-induced NOS activity and the NOS III phosphorylation at Ser1179 are puzzling. Consistent with our results is a recent study reporting that estrogen-induced NOS III phosphorylation was also dependent on MAPK activation (11). Therefore, it seems that MAPK can act indirectly to induce NOS III phosphorylation at Ser1179. Because the inhibition of PKA by H-89 did not alter MAPK activation induced by leptin, the prevention of both Ser1179 and Thr497 phosphorylation observed with MAPK inhibitors suggests that MAPK activation occurs upstream from PKA activation.

On the other hand, NOS III phosphorylation is under the control of protein phosphatases such as PP1 and PP2A (37) and, as recently shown, calcineurin (32). Using OA and tautomycin, we found in the present study results suggesting that PP1 activity, contrary to PP2A activity, is also required for leptin-induced NOS activity. PP1 is responsible for the dephosphorylation of NOS III at Thr497 (37). In contrast to the activation that occurs upon phosphorylation of Ser1179, phosphorylation of Thr497 alone interferes with the binding of calmodulin to NOS III, which results in attenuated NOS III activity (37). However, when Ser1179 and Thr497 are both phosphorylated, an increase in NOS activity can occur, as we observed herein with regard to leptin, DBcAMP, OA, or tautomycin alone and as previously shown with AMPK (13). Because inhibition of PP1 by tautomycin reduces NOS activity induced by leptin with a coordinated increase in Thr497 phosphorylation and a decrease in Ser1179 phosphorylation of NOS III, it can be suggested that PP1 reduces the Thr497 phosphorylation-inhibitory pathway, allowing Ser1179 phosphorylation. Insulin-induced PP1 activity is dependent on p42/p44 MAPK activation (3), and we recently showed that both PP1 and MAPK activities are necessary for the insulin-induced NO production in adipocytes (42). These findings led us to postulate that leptin-induced MAPK activation also could be indirectly involved in the NOS III phosphorylation at Ser1179 through PP1 control of Thr497 phosphorylation. Regardless of this hypothesis and the Akt and PKA pathways in the shear stress-induced NOS III activity, the respective roles taken by the MAPK and PKA activation pathways in leptin-induced NOS III activation remain to be clarified.

In conclusion, this study shows that leptin stimulates NOS activity in white adipocytes through a complex mechanism involving PKA, p42/p44 MAPK, and PP1. NO production by the adipocytes could explain the positive correlation between plasma nitrite levels and fat mass in rats (35) or in obese humans (14). NO is a signaling molecule involved in a critical range of processes, including vasodilatation, neurotransmission, endocrine signal transduction, immune defense, and angiogenesis. Previous studies have revealed that NO might be crucial in mediating the endocrine effects of leptin in pituitary gland and mononuclear cells (16, 52). Finally, the NO produced in response to leptin by adipocytes also could be a mediator of some leptin effects on adipose tissue metabolism, adipogenesis, or local angiogenesis.


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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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The experiments performed in our laboratory were supported by the Ministry of Research and Technology.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. Ribière, Dept. of Biochemistry and Molecular Biology, UFR Biomédicale des Saints-Pères, 75270 Paris Cedex 06, France (e-mail: catherine.ribiere{at}paris-ouest.univ-paris5.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.


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