No abstract

In isolated adipocytes, the nitrosothiols S-nitroso-Nacetyl-penicillamine (SNAP) and S-nitrosoglutathione stimulate basal lipolysis, whereas the nitric oxide (NO ⅐ ) 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 K m , 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 ␤-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 ⅐ is linked to both inhibition of the HSL activity and the cyclic AMP-dependent activation of HSL. These data suggest that NO ⅐ or related redox species like NO ؉ /NO ؊ are potential regulators of lipolysis through distinct mechanisms.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 lipo...

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 Ser(1179) and Thr(497), 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.

To elucidate the mechanisms through which 2-mercaptoacetate administration inhibits fatty acid oxidation in the liver, the respiration rates induced by different substrates were studied polarographically in rat hepatic mitochondria isolated 3 h after 2-mercaptoacetate administration. Palmitoyl-L-carnitine oxidation was almost completely inhibited in either the absence or presence of malonate. Octanoate oxidation was also inhibited, and the intramitochondrial acyl-CoA content was markedly increased. The oxidation rate of pyruvate and 2-oxoglutarate on the one hand and of 3-hydroxybutyrate, succinate and glutamate on the other was either normal or only slightly decreased. In the presence of 2,4-dinitrophenol, the extent of the inhibition of palmitoyl-L-carnitine oxidation was unchanged. All these results are consistent with the hypothesis that the 2-mercaptoacetate inhibition of fatty acid oxidation is due to an inhibition of the beta-oxidation pathway itself. Finally, the mitochondrial defect responsible for this inhibition was shown to be an inhibition of palmitoyl-CoA dehydrogenase activity (EC 1.3.99.3).