Rosa Señarís

Synthesis of Leptin in Human Placenta

Gender-based differences in serum leptin levels have been reported in umbilical cord blood, and leptin has been detected in human amniotic fluid. In order to understand if leptin may be directly synthesized by human placentae an analysis made up of several steps was performed. First at all RT-PCR analysis from placenta-derived RNA was used to detect human leptin mRNA. The leptin-like immunoradioactivity detected in placentae extracts was identical to human leptin according to the criteria of charge, immunorecognition, SDS-PAGE analysis and blotting, indicating that intact leptin was found and no variants in size, charge or immunoactivity were present in the placentae. Finally an immunohistochemical analysis showed the presence of leptin in the cytoplasm of syncytiotrophoblast cells but not in the core of villi. In conclusion: leptin is synthesized as a single molecular variant identical to human recombinant leptin in human placentae at delivery.

TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins.

Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

Gestational Profile of Leptin Messenger Ribonucleic Acid (mRNA) Content in the Placenta and Adipose Tissue in the Rat, and Regulation of the mRNA Levels of the Leptin Receptor Subtypes in the Hypothalamus During Pregnancy and Lactation

Abstract Serum leptin levels were significantly increased during rat gestation. Our data showed that leptin mRNA levels in both the adipose tissue and placenta were higher as pregnancy progressed, suggesting a role for both tissues in the hyperproduction of leptin. This paradoxical increase in leptin concentration during gestation suggests that a physiological state of leptin resistance may exist at the hypothalamic level that may explain the hyperphagia observed in pregnant rats. In order to study this issue further, levels of the mRNA encoding the different leptin receptor isoforms were determined in the hypothalamus of pregnant and nonpregnant rats. We found a specific reduction of the mRNA levels encoding the leptin receptor isoform Ob-Rb in the hypothalamus of pregnant rats compared to nonpregnant animals, suggesting that during pregnancy the hypothalamus shows a physiological resistance to the high levels of leptin due, at least in part, to a decrease in the expression of the long, biologically active form of the leptin receptor (Ob-Rb). During lactation, serum leptin levels returned to values observed in nonpregnant rats. In the hypothalami of these animals, Ob-Rb mRNA content was similar to that observed in nonpregnant rats, but we found an increased expression of some of the short forms of the leptin receptor (Ob-Re and Ob-Rf). This could contribute to induction of the hyperphagia present during lactation. These data provide new insights into the adaptive mechanisms that take place during pregnancy and lactation in order to meet increased metabolic requirements.

Hypothalamic levels of NPY, MCH, and prepro-orexin mRNA during pregnancy and lactation in the rat: role of prolactin

Pregnancy and lactation provide excellent models of physiological hyperphagia and hyper‐prolactinemia. To identify possible factors associated with the increased feeding in these situations, we measured hypothalamic mRNA levels of three orexigenic neuropeptides—NPY, MCH, and orexins—in nonpregnant, pregnant, and lactating rats by in situ hybridization. NPY mRNA content in the arcuate nucleus was significantly increased during pregnancy and lactation. However, MCH and prepro‐orexin expression was decreased in both states. 48 or 72 h of fasting in pregnant and lactating rats further elevated NPY mRNA levels and increased the low MCH mRNA content. Surprisingly, no effect was observed in prepro‐orexin mRNA levels. Finally, we investigated the possible effect of high PRL levels on these orexigenic signals using a model of hyperprolactinemia induced by pituitary graft. NPY mRNA content was unchanged, but MCH and prepro‐orexin mRNA levels were significantly decreased. Our results suggest that the increased NPY expression might be partly responsible for the hyperphagia observed during pregnancy and lactation. MCH and prepro‐orexin may be involved in the adaptation of other homeostatic mechanisms and their decreased levels in these physiological settings could be mediated by the elevated circulating PRL levels. García, M. C., López, M., Gualillo, O., Seoane, L. M., Diéguez, C., Señarís, R. M. Hypothalamic levels of NPY, MCH, and prepro‐orexin mRNA during pregnancy and lactation in the rat: role of prolactin. FASEB J. 17, 1392–1400 (2003)

Activation of growth hormone receptor delivers an antiapoptotic signal: evidence for a role of Akt in this pathway.

A signaling pathway was delineated by which GH promotes cell survival. Experiments were performed in human leukemic cells (HL-60) and Chinese hamster ovary (CHO) cells. In HL-60 cells, GH treatment reduced starvation-induced cell death. In contrast, when HL-60 cells were treated with an anti-GH antibody, cell survival was sharply reduced. In CHO cells stably expressing either the wild-type (wtGHR) or a truncated form (Δ454GHR) of the GH receptor in which GH induces a sustained activation of the receptor-associated tyrosine kinase JAK2, we found that GH stimulation inhibited programmed cell death induced by withdrawal of survival factors. This effect was enhanced in cells expressing the truncated form. In contrast, GH did not affect cell survival in CHO cells transfected with either the empty vector or a mutated GHR unable to transduce the signal (4P/AGHR). We also showed that the inhibitory action of GH on apoptosis is probably mediated via stimulation of the serine-threonine kinase Akt, as 1) GH treatmen...

Prolactin Stimulates Leptin Secretion by Rat White Adipose Tissue

Leptin, the obese (Ob) gene product, is an adipocyte-derived satiety factor that is involved in the regulation of food intake and body weight. Leptin signals nutritional status to several other physiological systems and modulates their function. As PRL is involved in energy and lipid metabolism, this study was undertaken to investigate the role of PRL on in vivo regulation of leptin serum concentration and Ob messenger RNA expression in white adipose tissue in rats. It was found that increased serum PRL levels, obtained by pituitary graft or exogenous injected ovine PRL (oPRL, 5 mg/kg), significantly stimulate serum leptin concentration. A significant increase (P < 0.01) in serum leptin concentration was present in hyperprolactinemic animals (4.7+/-0.4 microg/liter) in comparison to controls (1.2+/-0.1 microg/liter and 1.09+/-0.09 microg/liter of intact sham operated and ovariectomized rats, respectively). Similar results were obtained in oPRL-treated animals where leptin levels were 5.4+/-0.1 microg/liter vs. 1.1+/-0.1 microg/liter and 0.8+/-0.08 microg/liter of intact sham operated rats and ovariectomized, respectively (P < 0.001). This stimulatory effect of PRL on serum leptin levels was significantly reduced by food deprivation (P < 0.01) where serum leptin levels were 12.5+/-0.65 microg/liter in grafted animals vs. 3.2+/-0.36 microg/liter of grafted animals subjected to 48 h of food deprivation. Moreover, in vivo, PRL was able to induce leptin messenger RNA levels in several areas of rat white adipose tissue. The data demonstrate that PRL acts on the adipose tissue increasing leptin synthesis and secretion, suggesting a new role for this lactogenic hormone in the regulation of food intake.

Role of growth hormone (GH)-releasing hormone and somatostatin on leptin-induced GH secretion.

Leptin is a hormone secreted by the adipocytes that regulates food intake and energy expenditure. It is known that growth hormone (GH) secretion is markedly influenced by body weight, being suppressed in obesity and cachexia, and recent data have demonstrated that GH release is regulated by leptin levels. Although one of the sites of action of leptin is likely to be the hypothalamus, since leptin receptor mRNA is particularly abundant in several hypothalamic nuclei, the mechanisms by which leptin regulates GH secretion are not yet known. The aim of the present study was to investigate whether leptin could act at the hypothalamic level modulating somatostatin and GH-releasing hormone (GHRH) expression. The administration of anti-GHRH serum (500 µl, i.v.) completely blocked leptin-induced GH release in fasting rats. In contrast, the treatment with anti-somatostatin serum (500 µl, i.v.) significantly increased GH release in this condition. Furthermore, leptin administration (10 µg, i.c.v.) to intact fasting animals reversed the inhibitory effect produced by fasting on GHRH mRNA levels in the arcuate nucleus of the hypothalamus, and increased somatostatin mRNA content in the periventricular nucleus. Finally, leptin administration (10 µg, i.c.v.) to hypophysectomized fasting rats increased GHRH mRNA levels, and decreased somatostatin mRNA content, indicating an effect of leptin on hypothalamic GHRH- and somatostatin-producing neurons. These findings suggest a role for GHRH and somatostatin as mediators of leptin-induced GH secretion.

Sensing the fat: Fatty acid metabolism in the hypothalamus and the melanocortin system

Recent evidence has demonstrated that circulating long chain fatty acids act as nutrient abundance signals in the hypothalamus. Moreover, pharmacological inhibition of fatty acid synthase (FAS) results in profound decrease in food intake and body weight in rodents. These anorectic actions are mediated by the modulation of hypothalamic neuropeptide systems, such as melanocortins. In this review, we summarize what is known about lipid sensing and fatty acid metabolism in the hypothalamus. Understanding these molecular mechanisms could provide new pharmacological targets for the treatment of obesity and appetite disorders, as well as novel concepts in the nutritional design.

Neuropeptide Y, but Not Agouti-Related Peptide or Melanin-Concentrating Hormone, Is a Target Peptide for Orexin-A Feeding Actions in the Rat Hypothalamus

We examined the effects of orexin A on the mRNA levels of neuropeptide Y, agouti-related peptide, melanin-concentrating hormone, prepro-orexin and orexin receptors in the rat hypothalamus. Adult male rats were treated centrally (i.c.v.) with a single dose of orexin A (3 nmol). After 2, 6 and 12 h, neuropeptide Y, agouti-related peptide, melanin-concentrating hormone, and prepro-orexin mRNA levels were measured by semiquantitative RT-PCR and in situ hybridization; orexin receptors mRNA content was quantified by semiquantitative RT-PCR. We found that orexin A increased neuropeptide Y expression in the arcuate nucleus of the rat hypothalamus. This stimulatory effect was transient, being observed 2 h after the treatment, and disappearing after longer periods (6 and 12 h). In contrast, no change was demonstrated in hypothalamic agouti-related peptide, melanin-concentrating hormone, prepro-orexin or orexin receptors mRNA levels at any time evaluated. Our results suggest that neuropeptide Y synthesized in the arcuate nucleus of the hypothalamus, but not agouti-related peptide and melanin-concentrating hormone pathways, is likely involved in orexin-induced feeding behavior, and raise the possibility that this functional linkage may also be involved in other actions mediated by orexins such as locomotor activity and sympathetic function.

Effect of cyclic 3',5'-adenosine monophosphate, glucocorticoids, and insulin on leptin messenger RNA levels and leptin secretion in cultured human trophoblast.

Abstract Leptin is a polypeptide hormone originally thought to be produced exclusively by adipocytes. However, both leptin mRNA and leptin protein were identified in human placental trophoblast cells, suggesting a potential role in human pregnancy. In the present report, we examined the regulation of leptin mRNA levels and secretion by cAMP, glucocorticoids, and insulin in term human placental tissue. Placentae were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin concentrations were measured by ELISA in the cultured media of trophoblast maintained in monolayer culture for 24, 48, and 72 h. Likewise leptin mRNA levels in these cultured human trophoblast cells were determined by reverse transcription-polymerase chain reaction. Treatment with forskolin and (Bu)2 cAMP led to a time- and dose-dependent increase in leptin release, significant after 48 and 72 h. Moreover, incubation with forskolin for 48 h also clearly increased leptin mRNA concentration. Leptin secretion and mRNA levels were also assessed after treatment with insulin or dexamethasone. We found a time- and dose-dependent increase in leptin release, significant after 48 and 72 h. Leptin mRNA levels were also increased after these treatments. All this supports a stimulatory role of cAMP pathway, insulin and dexamethasone in the leptin mRNA levels, and leptin release in trophoblast cells in vitro.