Carmen González-Yanes

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.

The role of melatonin in the cells of the innate immunity: a review.

Melatonin is the major secretory product synthesized and secreted by the pineal gland and shows both a wide distribution within phylogenetically distant organisms from bacteria to humans and a great functional versatility. In recent years, a considerable amount of experimental evidence has accumulated showing a relationship between the nervous, endocrine, and immune systems. The molecular basis of the communication between these systems is the use of a common chemical language. In this framework, currently melatonin is considered one of the members of the neuroendocrine–immunological network. A number of in vivo and in vitro studies have documented that melatonin plays a fundamental role in neuroimmunomodulation. Based on the information published, it is clear that the majority of the present data in the literature relate to lymphocytes; thus, they have been rather thoroughly investigated, and several reviews have been published related to the mechanisms of action and the effects of melatonin on lymphocytes. However, few studies concerning the effects of melatonin on cells belonging to the innate immunity have been reported. Innate immunity provides the early line of defense against microbes and consists of both cellular and biochemical mechanisms. In this review, we have focused on the role of melatonin in the innate immunity. More specifically, we summarize the effects and action mechanisms of melatonin in the different cells that belong to or participate in the innate immunity, such as monocytes–macrophages, dendritic cells, neutrophils, eosinophils, basophils, mast cells, and natural killer cells.

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.

Role of Sam68 as an adaptor protein in signal transduction

Abstract.Sam68, the substrate of Src in mitosis, belongs to the family of RNA binding proteins. Sam68 contains consensus sequences to interact with other proteins via specific domains. Thus, Sam68 has various proline-rich sequences to interact with SH3 domain-containing proteins. Moreover, Sam68 also has a C-terminal domain rich in tyrosine residues that is a substrate for tyrosine kinases. Tyrosine phosphorylation of Sam68 promotes its interaction with SH2 containing proteins. The association of Sam68 with SH3 domain-containing proteins, and its tyrosine phosphorylation may negatively regulate its RNA binding activity. The presence of these consensus sequences to interact with different domains allows this protein to participate in signal transduction pathways triggered by tyrosine kinases. Thus, Sam68 participates in the signaling of T cell receptors, leptin and insulin receptors. In these systems Sam68 is tyrosine phosphorylated and recruited to specific signaling complexes. The participation of Sam68 in signaling suggests that it may function as an adaptor molecule, working as a dock to recruit other signaling molecules. Finally, the connection between this role of Sam68 in protein-protein interaction with RNA binding activity may connect signal transduction of tyrosine kinases with the regulation of RNA metabolism.

Metabolic effects and mechanism of action of the chromogranin A-derived peptide pancreastatin.

Pancreastatin is one of the regulatory peptides derived from intracellular and/or extracellular processing of chromogranin A, the soluble acidic protein present in the secretory granules of the neuroendocrine system. While the intracellular functions of chromogranin A include formation and maturation of the secretory granule, the major extracellular functions are generation of biologically active peptides with demonstrated autocrine, paracrine or endocrine activities. In this review, we will focus on the metabolic function of one of these peptides, pancreastatin, and the mechanisms underlying its effects. Many different reported effects have implicated PST in the modulation of energy metabolism, with a general counterregulatory effect to that of insulin. Pancreastatin induces glycogenolysis in liver and lipolysis in adipocytes. Metabolic effects have been confirmed in humans. Moreover, naturally occurring human variants have been found, one of which (Gly297Ser) occurs in the functionally important carboxy-terminus of the peptide, and substantially increases the peptides potency to inhibit cellular glucose uptake. Thus, qualitative hereditary alterations in pancreastatins primary structure may give rise to interindividual differences in glucose and lipid metabolism. Pancreastatin activates a receptor signaling system that belongs to the seven-spanning transmembrane receptor coupled to a Gq-PLCbeta-calcium-PKC signaling pathway. Increased pancreastatin plasma levels, correlating with catecholamines levels, have been found in insulin resistance states, such as gestational diabetes or essential hypertension. Pancreastatin plays important physiological role in potentiating the metabolic effects of catecholamines, and may also play a pathophysiological role in insulin resistance states with increased sympathetic activity.

Pancreastatin inhibits insulin action in rat adipocytes

Pancreastatin (PST), a regulatory peptide with a general inhibitory effect on secretion, is derived from chromogranin A, a glycoprotein present throughout the neuroendocrine system. We have previously demonstrated the counterregulatory role of PST on insulin action in rat hepatocytes. Here, we are reporting the PST effects on rat adipocytes. PST dose dependently inhibits basal and insulin-stimulated glucose transport, lactate production, and lipogenesis, impairing the main metabolic actions of insulin in adipocytes. These effects were observed in a wide range of insulin concentrations, leading to a shift to the right in the dose-response curve. Maximal effect was observed at 10 nM PST, and the IC50 value was approximately 1 nM. Moreover, PST has a lipolytic effect in rat adipocytes (ED50 0.1 nM), although it was completely inhibited by insulin. In contrast, PST dose dependently stimulated protein synthesis and enhanced insulin-stimulated protein synthesis. In summary, these data show the lipokinetic effect of PST and the inhibitory effect of PST on insulin metabolic action within a range of physiological concentrations. Therefore, these results give new pathophysiological basis for the association of PST with insulin resistance.

Pancreastatin, a chromogranin A-derived peptide, inhibits leptin and enhances UCP-2 expression in isolated rat adipocytes

Leptin, the ob gene product, is an adipocyte-secreted hormone that centrally regulates weight by decreasing caloric intake and increasing energy expenditure. Expression of leptin is regulated by dietary status, insulin, glucocorticoids and catecholamines. Pancreastatin (PST), a chromogranin A-derived peptide, correlates with catecholamine levels, and may play a role in the physiology of stress, modulating endocrine secretion and metabolism. Thus, PST has been found to exert a lipolytic and anti-insulin effect in white adipocytes. The aim of the present work was to investigate a possible role of PST modulating the expression of key genes involved in lipid storage and metabolism: leptin, PPAR-γ2, UCP-1 and UCP-2. We incubated isolated rat epididymal adipocytes with 100 nM PST for 16 and 24 h. Leptin, UCP-2 and UCP-1 mRNA levels were assessed by RT-PCR, followed by Southern blot. Leptin secretion was also measured by ELISA. PST inhibited leptin expression and secretion at 16-h incubation, but this effect was no longer observed after 24 h. On the other hand, PST stimulated the expression of UCP-2 after 16 h. However, the effect was still significant after 24 h. The inhibitory effect of PST on leptin expression and secretion and the stimulation of UCP-2 expression were prevented by blocking PKC. UCP-1 and PPR-γ2 expression did not change after PST stimulation. Leptin differentially regulates the expression of key genes in the rat adipocyte, upregulating the expression of UCP-2 and inhibiting the expression and secretion of leptin by a mechanism that involves PKC activity. These effects may contribute to the metabolic action of catecholamines in physiological and pathophysiological conditions with increased sympathetic activity.

Monitoring the Activation State of Insulin/Insulin-Like Growth Factor-1 Hybrid Receptors Using Bioluminescence Resonance Energy Transfer

In cells expressing both the insulin receptor isoform A (IRA) and the insulin-like growth factor-1 receptor (IGF1R), the presence of hybrid receptors, made up of an αβ-IRA chain associated with an αβ-IGF1R chain, has been demonstrated. These heterodimers are found in normal cells, and they also seem to play crucial roles in a number of cancers. However, they remain difficult to study, due to the concomitant presence of IRA and IGF1R homodimers. Using bioluminescence resonance energy transfer (BRET), we have developed assays to specifically monitor the activation state of IRA/IGF1R hybrids, both in vitro and in living cells. The first assay allowed the study of ligand-induced conformational changes within hybrid receptors purified from cells cotransfected with one type of receptor fused to Renilla reniformis luciferase (Rluc), and the other type of receptor fused to yellow fluorescent protein (YFP). In these conditions, only hybrid receptors were BRET-competent. In the second assay, the activation state of IRA/IGF1R hybrids was monitored in real time, in living cells, by cotransfection of kinase-dead versions of IRA-Rluc or IGF1R-Rluc, wild-type untagged IRA or IGF1R, and a YFP-tagged soluble version of the substrate-trapping mutant of protein tyrosine phosphatase 1B (YFP-PTP1B-D181A-Cter). In hybrid receptors, trans-phosphorylation of the kinase-dead αβ-Rluc moiety by the wild-type αβ moiety induced the recruitment of YFP-PTP1B-D181A-Cter, resulting in a hybrid-specific ligand-induced BRET signal. Therefore, both methods allow monitoring of the activity of IRA/IGF1R hybrid receptor and could be used to detect molecules of therapeutic interest for the treatment of cancer.

Characterization of pancreastatin receptors and signaling in adipocyte membranes

Pancreastatin (PST), a chromogranin A derived peptide with an array of effects in different tissues, has a role as a counterregulatory hormone of insulin action in hepatocytes and adipocytes, regulating glucose, lipid and protein metabolism. We have previously characterized PST receptors and signaling in rat hepatocytes, in which PST functions as a calcium-mobilizing hormone. In the present work we have studied PST receptors as well as the signal transduction pathways generated upon PST binding in adipocyte membranes. First, we have characterized PST receptors using radiolabeled PST as a ligand. Analysis of binding data indicated the existence of one class of binding sites, with a B(max) of 5 fmol/mg of protein and a K(d) of 1 nM. In addition, we have studied the G protein system that couples the PST receptor by gamma-(35)S-GTP binding studies. We have found that two G protein systems are involved, pertussis toxin-sensitive and -insensitive respectively. Specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Galpha(q/11) and to a lesser extent Galpha(i1,2) are activated by PST in rat adipocyte membranes. On the other hand, adenylate cyclase activity was not affected by PST. Finally, we have studied the specific phospholipase C isoform that is activated in response to PST. We have found that PST receptor is coupled to PLC-beta(3) via Galpha(q/11) activation in adipocyte membranes.