Víctor Sánchez-Margalet

Role of leptin as an immunomodulator of blood mononuclear cells: mechanisms of action

Leptin is a an adipocyte‐secreted hormone that regulates weight centrally. However, the leptin receptor is expressed not only in the central nervous system, but also in peripheral tissues, such as haematopoietic and immune systems. Therefore, the physiological role of leptin should not be limited to the regulation of food intake and energy expenditure. Moreover, the leptin receptor bears homology to members of the class I cytokine family, and recent data have demonstrated that leptin is able to modulate the immune response. Thus, the leptin receptor is expressed in human peripheral blood mononuclear cells, mediating the leptin effect on proliferation and activation. In vitro activation and HIV infection in vivo induce the expression of the long isoform of the leptin receptor in mononuclear cells. Also, leptin stimulates the production of proinflammatory cytokines from cultured monocytes and enhances the production of Th1 type cytokines from stimulated lymphocytes. Moreover, leptin has a trophic effect on monocytes, preventing apoptosis induced by serum deprivation. Leptin stimulation activates JAK–STAT, IRS‐1‐PI3K and MAPK signalling pathways. Leptin also stimulates Tyr‐phosphorylation of the RNA‐binding protein Sam68 mediating the dissociation from RNA. In this way, leptin signalling could modulate RNA metabolism. These signal transduction pathways provide possible mechanisms whereby leptin may modulate activation of peripheral blood mononuclear cells. Therefore, these data support the hypothesis regarding leptin as a proinflammatory cytokine with a possible role as a link between the nutritional status and the immune response. Moreover, these immunoregulatory functions of leptin could have some relevance in the pathophysiology of obesity.

Role of Leptin in the Activation of Immune Cells

Adipose tissue is an active endocrine organ that secretes various humoral factors (adipokines), and its shift to production of proinflammatory cytokines in obesity likely contributes to the low-level systemic inflammation that may be present in metabolic syndrome-associated chronic pathologies such as atherosclerosis. Leptin is one of the most important hormones secreted by adipocytes, with a variety of physiological roles related to the control of metabolism and energy homeostasis. One of these functions is the connection between nutritional status and immune competence. The adipocyte-derived hormone leptin has been shown to regulate the immune response, innate and adaptive response, both in normal and pathological conditions. The role of leptin in regulating immune response has been assessed in vitro as well as in clinical studies. It has been shown that conditions of reduced leptin production are associated with increased infection susceptibility. Conversely, immune-mediated disorders such as autoimmune diseases are associated with increased secretion of leptin and production of proinflammatory pathogenic cytokines. Thus, leptin is a mediator of the inflammatory response.

Leptin Promotes Cell Proliferation and Survival of Trophoblastic Cells

Abstract Leptin, the 16-kDa protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta. In the present work, we studied a possible effect of leptin on trophoblastic cell proliferation, survival, and apoptosis. Recombinant human leptin added to JEG-3 and BeWo choriocarcinoma cell lines showed a stimulatory effect on cell proliferation up to 3 and 2.4 times, respectively, measured by 3H-thymidine incorporation and cell counting. These effects were time and dose dependent. Maximal effect was achieved at 250 ng leptin/ml for JEG-3 cells and 50 ng leptin/ml for BeWo cells. Moreover, by inhibiting endogenous leptin expression with 2 μM of an antisense oligonucleotide (AS), cell proliferation was diminished. We analyzed cell population distribution during the different stages of cell cycle by fluorescence-activated cell sorting, and we found that leptin treatment displaced the cells towards a G2/M phase. We also found that leptin upregulated cyclin D1 expression, one of the key cell cycle-signaling proteins. Since proliferation and death processes are intimately related, the effect of leptin on cell apoptosis was investigated. Treatment with 2 μM leptin AS increased the number of apoptotic cells 60 times, as assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and the caspase-3 activity was increased more than 2 fold. This effect was prevented by the addition of 100 ng leptin/ml. In conclusion, we provide evidence that suggests that leptin is a trophic and mitogenic factor for trophoblastic cells by virtue of its inhibiting apoptosis and promoting proliferation.

Human leptin promotes survival of human circulating blood monocytes prone to apoptosis by activation of p42/44 MAPK pathway

Leptin, the adipocyte-secreted hormone, is known to function as an immunomodulatory regulator. Thus, we have recently found that human leptin promotes stimulation and proliferation of human peripheral blood mononuclear cells. Besides, we have also demonstrated that leptin triggers PI3K and p42/44 MAPK signaling pathways. In the present work, we sought to study the possible effect of leptin on cell survival and apoptosis, as well as the mechanisms underlying these effects. We have cultured human PBMC in serum-free conditions to assess the effect of leptin on cell survival and apoptosis. We have assayed the early phases of apoptosis by flow cytometric detection of phosphatidylserine expression using fluorescein isothiocyanate (FITC)-labelled Annexin V, simultaneously with dye exclusion of propidium iodide (PI), to discriminate intact cells, apoptotic, and necrotic cells. We have found that leptin promotes dose-dependent cell survival of monocytes after 24-96 h of serum-free culture. This effect of leptin on monocyte survival was completely reversed by blocking p42/44 MAPK activation employing the MEK inhibitor PD98059, whereas it was not affected by PI3K inhibition using Wortmannin. Leptin promotes this survival effect by preventing the apoptosis of monocyte cells, via MAPK activation. Thus, p42/44 MAPK inhibition, using PD98059, but not PI3K inhibition, employing Wortmannin, blocked the protective effect of leptin preventing apoptosis of monocytes cultured in the absence of serum. These data suggest that leptin is a trophic factor for the survival of blood monocytes and this effect is mediated by the p42/44 MAPK pathway.

Review: Leptin gene expression in the placenta ― Regulation of a key hormone in trophoblast proliferation and survival

Leptin is a 16000 MW protein originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblast cells. Study of the major signaling pathways known to be triggered by leptin receptor has revealed that leptin stimulates JAK/STAT, MAPK and PI3K pathways in placental cells. Leptin also exerts an antiapoptotic action in placenta and this effect is mediated by the MAPK pathway. Moreover, leptin stimulates protein synthesis by activating the translational machinery via both PI3K and MAPK pathways. Expression of leptin in placenta is highly regulated, suggesting that certain key pregnancy molecules participate in such regulation. An important hormone in reproduction, hCG, induces leptin expression in trophoblast cells and this effect involves the MAPK signal transduction pathway. Moreover, the cyclic nucleotide cAMP, which has profound actions upon human trophoblast function, also stimulates leptin expression and this effect seems to be mediated by crosstalk between the PKA and MAPK signaling pathways. Estrogens play a central role in reproduction. 17β-estradiol upregulates leptin expression in placental cells through genomic and non-genomic actions, probably via crosstalk between estrogen receptor-α and the MAPK and PI3K signal transduction pathways. Taken together these findings give a better understanding of the function of leptin and the regulatory mechanisms of leptin expression in human placental trophoblast and further support the importance of leptin in the biology of reproduction.

Elevated plasma total homocysteine levels in hyperinsulinemic obese subjects

Homocysteine has been associated with the oxidative stress in the pathogenesis of atherosclerosis. Oxidative stress caused by triglycerides and free fatty acids is known to cause insulin resistance and hyperinsulinemia. On the other hand, insulin resistance may increase homocysteine levels. Since obesity is associated with insulin resistance and hyperinsulinemia, we aimed to study the possible association of homocysteine with hyperinsulinemia in obese subjects. 20 obese male subjects (body mass index >29), aged 33--55 (mean 45 years old) were studied. A fasting blood sample was obtained for the study and the subjects undertook an oral glucose tolerance test with samples taken at 1 and 2 h after glucose. Subjects were divided in two groups according to the fasting insulin levels, < 9 &mgr;U/ml or normoinsulinemic (group 1) and >9 &mgr;U/ml or hyperinsulinemic (group 2). Glucose, insulin, homocysteine, folate, B(12,) total cholesterol, HDL-cholesterol and triglycerides levels were determined in fasting blood samples. In oral glucose tolerance test, glucose, insulin and homocysteine levels were measured. Hyperinsulinemic obese subjects (group 2) had higher levels of insulin and glucose at 1 h and 2 h postglucose, compared with group 1. Fasting total homocysteine and triglyceride levels were also increased in this group, whereas folate and B(12) levels were similar in both groups. Fasting homocysteine significantly correlated with fasting insulin (r = 0.6, p <0.01). Homocysteine levels slightly but significantly decreased after glucose loading in normoinsulinemic but not in hyperinsulinemic obese subjects. These results show that higher homocysteine levels are observed in the hyperinsulinemic obese subjects and suggest that homocysteine could play a role in the higher risk of cardiovascular disease in obesity.

17Beta-Estradiol Enhances Leptin Expression in Human Placental Cells Through Genomic and Nongenomic Actions

The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E2) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E2 effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E2-induced leptin expression. Moreover, E2 treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between −1951 and −1847 bp is both necessary and sufficient to achieve E2 effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E2 on leptin expression. Moreover, E2 action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E2 could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 μM PD98059 and 0.1 μM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E2 induction of leptin promoter. On the other hand, E2 treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E2 induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways.

Leptin prevents apoptosis of trophoblastic cells by activation of MAPK pathway

Leptin (Ob), the peripheral signal produced by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. Recently, we have demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work we aimed to study the signal transduction pathways that mediate the trophic effect of leptin in placenta, by using the human placenta choriocarcinoma JEG-3 cell line, as well as trophoblastic cells from human placenta. We have assayed the early phase of apoptosis, triggered by serum deprivation, by using Annexin V-propidium iodide (PI) labeling and flow cytometric analysis, as well as the late phase of apoptosis by studying the activation of caspase-3. We have studied the major signalling pathways known to be triggered by the leptin receptor, and we have investigated the relative importance of these pathways in the effect of leptin by using pharmacological inhibitors. We have found that leptin stimulates Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway by promoting JAK-2 and STAT-3 tyrosine phosphorylation. We have also demonstrated the activation of mitogen-activated protein kinase (MAPK) pathway by studying phosphorylation of extracellular-signal regulated kinase (Erk) kinase (MEK) and Erk1/2. PI3K pathway is also triggered by leptin stimulation as assessed by the study of protein kinase B (PKB) phosphorylation. These signaling pathways were confirmed in trophoblastic cells obtained from placenta of healthy donors. The effect of leptin on JEG-3 survival was completely reversed by blocking Erk1/2 activation employing the MEK inhibitor PD98059, whereas it was not affected by PI3K inhibition using wortmannin. These data suggest that the leptin antiapoptotic effect in placenta is mediated by the MAPK pathway.

Leptin receptor (Ob-R) expression is induced in peripheral blood mononuclear cells by in vitro activation and in vivo in HIV-infected patients

Leptin, the Ob gene product, is an adipocyte hormone that centrally regulates weight control. In addition, other effects of leptin in peripheral tissues have been described. Thus, leptin has been found to regulate reproduction, haematopoiesis and immune function. We have found recently that leptin has a stimulatory effect on human peripheral blood mononuclear cells (PBMC). Monocytes are activated by leptin alone whereas T lymphocytes need a suboptimal stimulus of PHA or ConA before further activation by leptin. These effects are mediated by the long isoform of the leptin receptor, which has been shown to trigger signalling in PBMC. In fact, we have found that human leptin stimulates Janus kinase (JAK)‐signal transducer and activator of transcription (STAT), phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) pathways in PBMC. In order to assess possible regulation of the long isoform of the leptin receptor (Ob‐R) in mononuclear cells upon activation, we have studied the expression of Ob‐R by RT‐PCR and Western blotting in PBMC activated in vitro by PHA or ConA and in vivo in HIV‐infected patients. We have found that in vitro activation and in vivo HIV infection correlates with an increase in leptin receptor expression in PBMC. Moreover, the leptin receptor is tyrosine phosphorylated in PBMC from HIV‐infected patients, suggesting that the leptin receptor is activated. These results are consistent with the suggested role of leptin in modulating the immune response.

Up-Regulation of Placental Leptin by Human Chorionic Gonadotropin

Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway.