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.

Nongenomic estrogen effects on nitric oxide synthase activity in rat adipocytes.

Estrogens exert multiple genomic effects on adipose tissue through binding to nuclear estrogen receptors. However, there is evidence for additional nongenomic mechanisms whereby estrogens may exert their control on adipose tissue metabolism through rapid activation of various membrane-initiated kinase cascades. Here, we tested rapid effects of estrogens on nitric oxide production in white adipose tissue using 17-beta estradiol (E2) and its membrane impermeant albumin conjugated form (17-beta estradiol hemisuccinate BSA, E2-BSA). We found that both E2 and E2-BSA stimulate nitric oxide synthase (NOS) activity in adipocytes. These effects were abolished by 1) ICI 182-780, a selective estrogen receptor antagonist; 2) wortmannin, an inhibitor of phosphatidylinositol 3-kinase; and 3) N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89) an inhibitor of protein kinase A. In contrast to NOS activation by E2, E2-BSA-induced NOS activity was abolished by UO126, an inhibitor of MAPK kinase/ERK (p42/p44 MAPKs). Immunoblotting studies have shown that both estrogens phosphorylate endothelial NOS (NOS III) on Ser(1179), an effect that is prevented by wortmannin and H89, suggesting that NOS III is the target for estrogen-induced NOS activity. Furthermore, only the E2-BSA-induced NOS III phosphorylation on Ser(1179) was totally abolished by UO126. These results indicate that the signaling cascades involved in adipocyte NOS stimulation by estrogens are different depending on whether estrogens are free or conjugated to albumin and therefore underline the importance of estrogen receptor locations in the nongenomic actions of estrogens in these cells.

Gender difference in diet-induced obesity hypertension: implication of renal alpha2-adrenergic receptors.

Although the pathogenesis of the obesity-related hypertension is not fully understood, prevalence of the cardiovascular complications is much higher in obese men than obese women. In a recent study, we reported that male rats fed a cafeteria diet, while becoming obese, developed hypertension and important changes in their renal alpha2-adrenergic receptor subtypes distributions. The aim of the present study was to investigate whether these alterations are sex dependent. After 10 weeks of the cafeteria diet, male and female rats had the same increase in fat pad weight and in plasma leptin levels. However, in contrast to males, females had normal blood pressure (BP). On the basis of radioligand-binding studies using [3H]-RX821002 and confirming our recent observation, an increase in alpha2-adrenergic receptor densities occurred in kidneys of cafeteria-fed male but not female rats. Moreover, in contrast with the situation observed in males, ligand competition studies failed to reveal any change in the renal alpha2A- and alpha2B-adrenergic receptors subtypes distribution in females. Finally, in the cafeteria-fed females reverse transcription-polymerase chain reaction experiments showed unaltered expression of these two alpha2-adrenergic receptor subtypes. These data thus suggest a strong relationship between the sexual dimorphism in the cafeteria diet-induced hypertension and altered expression of the alpha2-adrenergic receptor subtypes in the kidney.

Pesticide adsorption in the vadose zone: a case study on Eocene and Quaternary materials in Northern France.

We present a set of adsorption coefficients measured on various Eocene and Quaternary materials sampled from the vadose zone of a catchment in Northern France for three herbicides, atrazine, isoproturon and metamitron. Some vadose zone materials were found to have higher adsorption coefficients than the topsoil. The adsorption coefficients were strongly dependent on the clay content of the material. From 83% to 97% of the variability in the adsorption coefficients could be explained by a linear relationship to the clay content. Adsorption coefficients normalized for clay content, Kclay, ranged between 1.6 and 17.6 litre kg(-1) for atrazine. Neglecting the adsorption properties of the vadose zone and relying exclusively on Koc values to predict mobility may bias regional or local risk assessment of groundwater contamination by pesticides. More information on the adsorption properties of geological materials should be collected to improve our ability to predict pesticide concentrations in groundwaters.

Nutritional regulation of leptin signaling.

The adipose-derived hormone leptin was first described as a satiety factor, but recent studies have demonstrated that leptin acts on various physiologic processes and plays an important role in obesity and the associated hypertension. In this article, we review recent data on leptin signaling as it relates to nutrition. Plasma leptin levels are positively correlated to body fat and adipocyte size and, therefore, levels are higher during obesity. The hyperphagia in the presence of hyperleptinemia in obesity is a paradoxical effect. Leptin signaling primarily depends on the leptin receptor (Ob-R). The suppressor of cytokine-signaling (SOCS) protein, in particular SOCS-3, was shown as a leptin-regulated inhibitor of proximal leptin signaling, although its role during obesity remains uncertain.

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

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.

Factors that influence the risk of hypertension in obese individuals.

PURPOSE OF REVIEW: Obesity, which has reached epidemic prevalence, is now recognized as an independent risk factor for increasing blood pressure. The complex mechanisms of obesity-related hypertension are unclear, but several studies have provided evidence of a hypertensive shift in pressure natriuresis. Excess sympathetic outflow to the kidneys and changes in renal structure and function may both affect the renal pressure relationship. Other factors that may contribute to altered natriuresis include hyperinsulinemia, hyperleptinemia and activation of the renin-angiotensin system. Disruption of the renal alpha2 adrenoceptors or leptin receptor implicated in natriuresis control may also be an additive risk for the increase in tubular reabsorption in obesity hypertension. RECENT FINDINGS: Recent advances have highlighted the importance of two adipocyte-derived hormones - leptin and angiotensinogen - in obesity hypertension. Leptin has direct central effects that increase sympathetic outflow to the kidney and the new concept of selective leptin resistance, suggests the maintenance of leptin-induced sympathetic activation in obesity, despite resistance to leptin metabolic effects. On the other hand, a recent study showed that angiotensinogen produced in the adipocyte is also relevant to blood pressure control. SUMMARY: The article reviews the factors implicated in the disruption of blood pressure control in obesity. Further investigation on the time course of the disease would reveal the relative importance of each of the factors that influence the risk of hypertension in obese individuals.

Hypothalamic leptin receptor and signaling molecule expressions in cafeteria diet-fed rats.

Although obesity is associated with a state of leptin resistance, it has been suggested that leptin may contribute to the pathogenesis of obesity-related hypertension. In previous studies, we reported that cafeteria diet feeding induces hyperleptinaemia and hyperinsulinemia in both male and female rats, with hypertension occurring only in male rats. However, when female rats were neonatally treated with testosterone (T), these animals develop hypertension when fed the cafeteria diet. These observations led us to investigate leptin signaling and some neuropeptides that are leptin targets in the hypothalamus of male, intact female, and T-treated female cafeteria diet-fed rats. A decrease in the hypothalamic leptin receptors (Ob-Ra and Ob-Rb) and pro-opiomelanocortin (POMC) mRNA was observed only in male hypertensive cafeteria diet-fed rats. Although no alterations in Ob-R occurred in both groups of female cafeteria diet-fed rats, the hyperleptinaemic state of these animals had no influence on POMC mRNA levels. In intact female rats, expression of the suppressors of cytokines signaling SOCS-1, SOCS-2, SOCS-3, and cytokine inhibitor signaling were unaltered, whereas in T-treated females SOCS-3 was overexpressed. Finally SOCS-1 mRNA level was increased only in male rats. Because hyperinsulinemia was reported to counteract the leptin-induced stimulation of the sympathetic tone and because SOCS-1 and -3 are potential inhibitors of insulin signaling, our results suggest that the hypothalamic overexpression of SOCS-1 or SOCS-3 found in male or T-treated female rats after cafeteria diet feeding could block the negative influence of the hyperinsulinemia on the central pressor action of leptin, thereby contributing to their hypertensive state.

Gender differences in hypothalamic tyrosine hydroxylase and alpha(2)-adrenoceptor subtype gene expression in cafeteria diet-induced hypertension and consequences of neonatal androgenization.

This study investigated the incidence of cafeteria-diet induced hypertension on hypothalamic tyrosine hydroxylase (TH) and alpha(2)-adrenoceptor subtype gene expression in male, female, and neonatally testosterone-imprinted female rats. After 10 weeks of cafeteria diet, all these rats were hyperleptinemic. In contrast, males and testosterone-treated females developed hypertension, whereas intact females remained normotensive. In these rats, cafeteria diet up-regulated TH gene expression only in males and testosterone-treated females. On the other hand, cafeteria diet differentially affected hypothalamic gene expression of alpha(2)-adrenoceptor subtypes. In fact, this diet increased alpha(2A)-adrenoceptor mRNA levels only in intact normotensive females. In contrast, gene expression of the alpha(2B)-adrenoceptor was up-regulated only in male and testosterone-treated female cafeteria-fed rats. Furthermore, an alpha(2C)-adrenoceptor gene over-expression was also induced, but only in male cafeteria-fed rats. If one assumes that the up-regulations in TH and alpha(2B)-adrenoceptor gene expression are indicative of increased sympathetic nervous activity, then, these altered gene expressions could be responsible for the maintenance of high blood pressure in male and testosterone-treated female cafeteria-fed rats. Conversely, in intact females, the absence of these over-expressions and the up-regulation of the alpha(2A)-adrenoceptor gene expression could reflect an adaptive response to the diet and, consequently, could be protective against cafeteria diet-induced hypertension. Moreover, neonatal testosterone imprinting in females could have induced an irreversible android susceptibility to the cafeteria diet, leading to the onset of hypertension.

Sexual dimorphism in cafeteria diet-induced hypertension is associated with gender-related difference in renal leptin receptor down-regulation.

Plasma leptin levels are elevated in obesity suggesting a pathophysiologic role of this hormone in obesity and related disorders, such as hypertension. Furthermore, despite excess leptin levels, leptin satiety action is blunted in obesity suggesting the occurrence of central leptin resistance. As leptin acts on the kidney to induce natriuresis, renal leptin receptor alterations could lead to a defect in sodium excretion and hence to hypertension. Therefore, the present study investigated renal leptin receptor (Ob-Ra and Ob-Rb) mRNA and leptin binding capacities in diet-induced hypertension. Feeding male, female, and testosterone-treated female rats a cafeteria diet for 10 weeks increased body fat mass, plasma insulin, and leptin levels. Furthermore, although male and testosterone-treated female cafeteria-fed rats were hypertensive, the female rats fed the same diet failed to develop elevated blood pressure. In renal medulla, Ob-Ra and Ob-Rb mRNA levels were unchanged after cafeteria diet feeding in all groups; however, binding analysis revealed Ob-R protein down-regulation exclusively in hypertensive rats. Moreover, renal Ob-R densities were inversely correlated to plasma leptin concentrations in male rats and testosterone-treated female rats but not in female rats. These findings demonstrate the existence of differences in renal Ob-R binding capacities, which are correlated to hypertension.

Insulin stimulates nitric oxide production in rat adipocytes.

In adipocytes, insulin regulates the activity of different protein kinases (PI3K/Akt, MAPK, PKC) and protein phosphatases (PP-1, PP-2A). Since these enzymes are implicated in the regulation of NOS activity which is present in adipose tissue, we tested the effects of insulin on white adipocyte NOS activity. Exposure of adipocytes to insulin resulted simultaneously in NOS activity stimulation and Akt activation with maximal effect observed at 1 nM. Higher concentrations of insulin induced a progressive decline of NOS activity. In the presence of wortmannin, a PI3K inhibitor, 1 nM insulin failed to stimulate NOS activity. Insulin (1 nM)-stimulated NOS activity was also abolished by U0126, an inhibitor of p42/p44 MAPK activation, and by 1 microM okadaic acid (OA), which inhibits both PP-1 and PP-2A but not by 1 nM OA which inhibits only PP-2A. Moreover, inhibition of cPKC allowed a high (1 microM) insulin concentration to stimulate NOS activity. These results (i) demonstrate that insulin activates NO production in adipocytes through both PI3K/Akt and MAPK/PP-1 activation and (ii) suggest that PP-1 activation protects NOS against the inhibitory effect of cPKC activation.