Leptin induces nitric oxide-mediated inhibition of lipolysis and glyceroneogenesis in rat white adipose tissue.

Leptin is secreted by white adipose tissue (WAT) and induces lipolysis and nonesterified fatty acid (NEFA) oxidation. During lipolysis, NEFA efflux is the result of triglyceride breakdown, NEFA oxidation, and re-esterification via glyceroneogenesis. Leptin's effects on glyceroneogenesis remain unexplored. We investigated the effect of a long-term treatment with leptin at a physiological concentration (10 µg/L) on lipolysis and glyceroneogenesis in WAT explants and analyzed the underlying mechanisms. Exposure of rat WAT explants to leptin for 2 h resulted in increased NEFA and glycerol efflux. However, a longer treatment with leptin (18 h) did not affect NEFA release and reduced glycerol output. RT-qPCR showed that leptin significantly downregulated the hormone-sensitive lipase (HSL), cytosolic phosphoenolpyruvate carboxykinase (Pck1), and PPARγ genes. In agreement with its effect on mRNA, leptin also decreased the levels of PEPCK-C and HSL proteins. Glyceroneogenesis, monitored by [1-(14) C] pyruvate incorporation into lipids, was reduced. Because leptin increases nitric oxide (NO) production in adipocytes, we explored the role of NO in the leptin signaling pathway. Pretreatment of explants with the NO synthase inhibitor Nω-nitro-l-arginine methyl ester eliminated the effect of leptin on lipolysis, glyceroneogenesis, and expression of the HSL, Pck1, and PPARγ genes. The NO donor S-nitroso-N-acetyl-DL penicillamine mimicked leptin effects, thus demonstrating the role of NO in these pathways. The inverse time-dependent action of leptin on WAT is consistent with a process that limits NEFA re-esterification and energy storage while reducing glycerol release, thus preventing hypertriglyceridemia.

Leptin and insulin induce mutual resistance for nitric oxide synthase III activation in adipocytes.

Obesity-induced hyperleptinemia is frequently associated with insulin resistance suggesting a crosstalk between leptin and insulin signaling pathways. Our aim was to determine whether insulin and leptin together interfere on NOS activation in adipocytes. We examined insulin and leptin-induced nitric oxide synthase (NOS) activity, protein amount and NOS III phosphorylation at Ser(1179) in isolated epididymal adipocytes from rat, in the presence or not of inhibitors of kinases implicated in insulin or leptin signaling pathways. Insulin or leptin induced NOS III phosphorylation at Ser(1179) leading to increased NO production in rat adipocytes, in agreement with our previous observations. When insulin and leptin at a concentration found in obese rats (10 ng/ml) were combined, NOS activity was not increased, suggesting a negative crosstalk between insulin and leptin signaling mechanisms. Chemical inhibitors of kinases implicated in signaling pathways of insulin, such as PI-3 kinase, or of leptin, such as JAK-2, did not prevent this negative interaction. When leptin signaling was blocked by PKA inhibitors, insulin-induced NOS activity and NOS III phosphorylation at Ser(1179) was observed. In the presence of leptin and insulin, (i) IRS-1 was phosphorylated on Ser(307) and this effect was prevented by PKA inhibitors, (ii) JAK-2 was dephosphorylated, an effect prevented by SHP-1 inhibitor. A mutual resistance occurs with leptin and insulin. Leptin phosphorylates IRS-1 to induce insulin resistance while insulin dephosphorylates JAK-2 to favor leptin resistance. This interference between insulin and leptin signaling could play a crucial role in insulin- and leptin-resistance correlated with obesity.

Mycolactone subverts immunity by selectively blocking the Sec61 translocon.

Mycolactone, an immunosuppressive macrolide released by the human pathogen Mycobacterium ulcerans, was previously shown to impair Sec61-dependent protein translocation, but the underlying molecular mechanism was not identified. In this study, we show that mycolactone directly targets the α subunit of the Sec61 translocon to block the production of secreted and integral membrane proteins with high potency. We identify a single-amino acid mutation conferring resistance to mycolactone, which localizes its interaction site near the lumenal plug of Sec61α. Quantitative proteomics reveals that during T cell activation, mycolactone-mediated Sec61 blockade affects a selective subset of secretory proteins including key signal-transmitting receptors and adhesion molecules. Expression of mutant Sec61α in mycolactone-treated T cells rescued their homing potential and effector functions. Furthermore, when expressed in macrophages, the mycolactone-resistant mutant restored IFN-γ receptor-mediated antimicrobial responses. Thus, our data provide definitive genetic evidence that Sec61 is the host receptor mediating the diverse immunomodulatory effects of mycolactone and identify Sec61 as a novel regulator of immune cell functions.

Metabolomic profiles delineate mycolactone signature in Buruli ulcer disease.

Infection of human skin with Mycobacterium ulcerans, the causative agent of Buruli ulcer, is associated with the systemic diffusion of a bacterial macrolide named mycolactone. Patients with progressive disease show alterations in their serum proteome, likely reflecting the inhibition of secreted protein production by mycolactone at the cellular level. Here, we used semi-quantitative metabolomics to characterize metabolic perturbations in serum samples of infected individuals, and human cells exposed to mycolactone. Among the 430 metabolites profiled across 20 patients and 20 healthy endemic controls, there were significant differences in the serum levels of hexoses, steroid hormones, acylcarnitines, purine, heme, bile acids, riboflavin and lysolipids. In parallel, analysis of 292 metabolites in human T cells treated or not with mycolactone showed alterations in hexoses, lysolipids and purine catabolites. Together, these data demonstrate that M. ulcerans infection causes systemic perturbations in the serum metabolome that can be ascribed to mycolactone. Of particular importance to Buruli ulcer pathogenesis is that changes in blood sugar homeostasis in infected patients are mirrored by alterations in hexose metabolism in mycolactone-exposed cells.

Combined inflammatory and metabolic defects reflected by reduced serum protein levels in patients with Buruli ulcer disease.

Buruli ulcer is a skin disease caused by Mycobacterium ulcerans that is spreading in tropical countries, with major public health and economic implications in West Africa. Multi-analyte profiling of serum proteins in patients and endemic controls revealed that Buruli ulcer disease down-regulates the circulating levels of a large array of inflammatory mediators, without impacting on the leukocyte composition of peripheral blood. Notably, several proteins contributing to acute phase reaction, lipid metabolism, coagulation and tissue remodelling were also impacted. Their down-regulation was selective and persisted after the elimination of bacteria with antibiotic therapy. It involved proteins with various functions and origins, suggesting that M. ulcerans infection causes global and chronic defects in the host's protein metabolism. Accordingly, patients had reduced levels of total serum proteins and blood urea, in the absence of signs of malnutrition, or functional failure of liver or kidney. Interestingly, slow healers had deeper metabolic and coagulation defects at the start of antibiotic therapy. In addition to providing novel insight into Buruli ulcer pathogenesis, our study therefore identifies a unique proteomic signature for this disease.

Is there NO help for leptin?

Since the initial identification of leptin as the product of the ob gene in 1994, the signaling pathways by which this hormone alters cell physiology have been the subject of extensive investigations. The fact that leptin can induce nitric oxide (NO) production was first demonstrated in studies of the pituitary gland and pancreatic islets. A large number of additional studies further showed that this adipokine stimulates NO synthesis in multiple tissues. This review article discusses the role of leptin in NO production and its pathophysiological consequences. The role of this gaseous messenger in cell physiology depends on the cell type, the concentration of NO and the duration of exposure. It can be either a potent oxidant or a protector of cell integrity against the formation of reactive oxygen species. Leptin plays two opposing roles on arterial pressure. It exerts a hypertensive effect due to sympathetic activation and a vasorelaxant effect due to NO production. This adipokine acts via NO to produce pro-inflammatory factors in cartilage pathology, potentially contributing to an increased risk for osteoarthritis. Another well-documented role of leptin-induced NO, acting either directly or via the hypothalamus, concerns lipid metabolism in muscle and adipose tissue. In adipocytes, the direct and rapid action of leptin is to activate the nitric oxide synthase III, which favors lipolysis. In contrast, in the long-term, leptin reduces lipolysis. However, both in the short-term and in the long-term, glyceroneogenesis and its key enzyme, the cytosolic phosphoenolpyruvatecarboxykinase (PEPCK-C), are down-regulated by the adipokine, thus favoring fatty acid release. Hence, leptin-induced NO production plays a crucial role in fatty acid metabolism in adipose tissue. The resulting effects are to prevent lipid storage and to improve energy expenditure, with possible improvements of the obese state and its associated diseases.

Rapid nitration of adipocyte phosphoenolpyruvate carboxykinase by leptin reduces glyceroneogenesis and induces fatty acid release.

Fatty acid (FA) release from white adipose tissue (WAT) is the result of the balance between triglyceride breakdown and FA re-esterification. The latter relies on the induction of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), the key enzyme for glyceroneogenesis. We previously demonstrated that long-term (18 h) leptin treatment of rat epididymal WAT explants reduced glyceroneogenesis through nitric oxide (NO)-induced decrease in PEPCK-C expression. We investigated the effect of a short-term leptin treatment (2 h) on PEPCK-C expression and glyceroneogenesis in relation to NO production. We demonstrate that in WAT explants, leptin-induced NO synthase III (NOS III) phosphorylation was associated with reduced PEPCK-C level and glyceroneogenesis, leading to FA release, while PEPCK-C gene expression remained unaffected. These effects were absent in WAT explants from leptin receptor-deficient Zucker rat. Immunoprecipitation and western blot experiments showed that the leptin-induced decrease in PEPCK-C level was correlated with an increase in PEPCK-C nitration. All these effects were abolished by the NOS inhibitor Nω-nitro-L-arginine methyl ester and mimicked by the NO donor S-nitroso-N-acetyl-DL penicillamine. We propose a mechanism in which leptin activates NOS III and induces NO that nitrates PEPCK-C to reduce its level and glyceroneogenesis, therefore limiting FA re-esterification in WAT.