R. Señaris

Regulation of in Vivo Growth Hormone Secretion by Leptin

Leptin, the product of the ob gene, is a recently discovered hormone secreted by adipocytes that regulates food intake and energy expenditure. Growth hormone (GH) secretion is markedly influenced by body weight being markedly suppressed in obesity and underweight. The aim of the present study was to study whether leptin can act as a metabolic signal connecting the adipose tissue with the growth hormone axis. We administered leptin antiserum (10 ul, i.c.v.) or normal rabbit serum (NRS; 10 ul, i.c.v.) to freely moving fed rats. Furthermore we assessed the effect of leptin administration (10 ug, i.c.v.) on fed and fasted rats. Spontaneous GH secretion was assessed over 6 hours with blood samples taken every 15 min. Administration of leptin antiserum led to a decrease in spontaneous GH secretion as assessed by the area under the curve (AUC) (168 ± 72 ng/ml/6h) in comparison to NRS-treated rats (813 ± 179 ng/ml/6 h, p < 0.01). While leptin administration (10 ug/rat; i.c.v.) to normal fed rats did not modify sp...

Orexin Receptors Are Expressed in the Adrenal Medulla of the Rat

Two recently discovered hypothalamic peptides, orexin-A and orexin-B, play a role as mediators in the central mechanisms that regulate feeding behavior and sleep control. These peptides bind and activate two orexin receptors that belong to the G-protein coupled receptor superfamily. Morphological studies have detected mRNA expression of orexin receptors exclusively in the rat central nervous system. In this paper we demonstrate a strong level of expression of orexin receptor 1 and 2 in the adrenal medulla of the rat by RT-PCR immunohistochemistry. The results of the present study provide the first evidence showing that the adrenal medulla expresses orexin receptors, and thus appears to be a target tissue for orexins. This could open a new loop in which the central and autonomous nervous system may be involved in body weight homeostasis and sleep control.

A possible role of neuropeptide Y, agouti-related protein and leptin receptor isoforms in hypothalamic programming by perinatal feeding in the rat.

Aim/hypothesisPerinatal overfeeding predisposes humans and rats to obesity and diabetes in later life. One classical model for studying the effect of early feeding is manipulation of the size of rat litters. Rats growing up in small litters gain more weight than rats growing up in normal-sized litters. Interestingly, these obese rats maintain this phenotype in adulthood. Conversely, rats raised in large litters show a delay in growth and a decrease in body weight. The aim of this work was to assess the hypothalamic control mechanisms of food intake regulated by perinatal feeding.MethodsLeptin levels were analysed using RIA. Leptin receptor mRNA levels were analysed using RT-PCR. Neuropeptide mRNA levels were analysed using in situ hybridisation.ResultsPerinatally overfed neonatal male rats exhibited hyperleptinaemia and a decrease in hypothalamic mRNA levels of the long isoform of the leptin receptor (OB-Rb), explaining their leptin resistance. Moreover, this obese model showed an increase in the mRNA expression of cocaine- and amphetamine-regulated transcript, neuropeptide Y and agouti-related protein in the hypothalamic arcuate nucleus (ARC). In contrast, perinatally underfed neonatal male rats with hypoleptinaemia showed an increase in hypothalamic mRNA of the short isoforms of the leptin receptor. Furthermore, they exhibited an increase in expression of neuropeptide Y and agouti-related protein in the ARC.Conclusions/interpretationRats overfed during early postnatal life show a leptin-resistant state mediated by down-regulation of the hypothalamic OB-Rb. These data, together with the increased expression of neuropeptide Y and agouti-related protein in specific neurons in the ARC, might indicate the existence of regulated programming in this nucleus and may provide a new aetiopathogenic concept in susceptibility to obesity.

Leptin Inhibits in Vitro Hypothalamic Somatostatin Secretion and Somatostatin mRNA Levels

Leptin, the product of the ob gene, is a recently discovered hormone secreted by adipocytes that regulates food intake and energy expenditure. The site of action of leptin is likely to be the hypothalamus, since this area is important in the control of food intake and leptin receptor mRNA is particularly abundant in this area. In order to further unravel the mechanisms by which leptin acts, we have studied the effect of leptin on in vitro somatostatin synthesis and secretion. Leptin administration to fetal rat neurones in monolayer culture led to a time dependent decrease in basal somatostatin secretion and somatostatin mRNA levels, the maximal effect being observed with 6x10(-8) M leptin after 24 h incubation. Furthermore, leptin completely blunted 10(-7) M Neuropeptide Y-induced increase in somatostatin secretion and somatostatin mRNA levels as well as 10(-3) M (Bu)2-cAMP and 10(-6) M A23187-induced somatostatin secretion. Finally, leptin (3x10(-8) M M) also inhibited low glucose (1.1 mM) induced-somatostatin secretion in perifused adult hypothalami. This data indicates that leptin can influence the neuroendocrine system by regulating hypothalamic somatostatin gene expression.

Hyperphagia and Central Mechanisms for Leptin Resistance during Pregnancy

The purpose of this work was to study the central mechanisms involved in food intake regulation and leptin resistance during gestation in the rat. Sprague Dawley rats of 7, 13, and 18 d of pregnancy [days of gestation (G) 7, G13, and G18] were used and compared with nonpregnant animals in diestrus-1. Food intake was already increased in G7, before hyperleptinemia and central leptin resistance was established in midpregnancy. Leptin resistance was due to a reduction in leptin transport through the blood-brain barrier (BBB) and to alterations in leptin signaling within the hypothalamus based on an increase in suppressor of cytokine signaling 3 levels and a blockade of signal transducer and activator of transcription-3 phosphorylation (G13), followed by a decrease in LepRb and of Akt phosphorylation (G18). In early gestation (G7), no change in hypothalamic neuropeptide Y (NPY), agouti-related peptide (AgRP), or proopiomelanocortin (POMC) expression was shown. Nevertheless, an increase in NPY and AgRP and a decrease in POMC mRNA were observed in G13 and G18 rats, probably reflecting the leptin resistance. To investigate the effect of maternal vs. placental hormones on these mechanisms, we used a model of pseudogestation. Rats of 9 d of pseudogestation were hyperphagic, showing an increase in body and adipose tissue weight, normoleptinemia, and normal responses to iv/intracerebroventricular leptin on hypothalamic leptin signaling, food intake, and body weight. Leptin transport through the BBB, and hypothalamic NPY, AgRP and POMC expression were unchanged. Finally, the transport of leptin through the BBB was assessed using a double-chamber culture system of choroid plexus epithelial cells or brain microvascular endothelial cells. We found that sustained high levels of prolactin significantly reduced leptin translocation through the barrier, whereas progesterone and β-estradiol did not show any effect. Our data demonstrate a dual mechanism of leptin resistance during mid/late-pregnancy, which is not due to maternal hormones and which allows the maintenance of hyperphagia in the presence of hyperleptinemia driven by an increase in NPY and AgRP and a decrease in POMC mRNA. By contrast, in early pregnancy maternal hormones induce hyperphagia without the regulation of hypothalamic NPY, AgRP, or POMC and in the absence of leptin resistance.

Prepro-orexin mRNA levels in the rat hypothalamus, and orexin receptors mRNA levels in the rat hypothalamus and adrenal gland are not influenced by the thyroid status

Orexins are two recently discovered neuropeptides that play an important role in the regulation of food intake and in the regulation of the sleep-wake cycle. In this work we examined the effects of thyroid hormones on prepro-OX expression in the rat hypothalamus, and OXRs expression in the rat hypothalamus and adrenal gland. Hypo- and hyperthyroidism were induced in adult male rats, and the levels of hypothalamic prepro-OX and OXRs mRNA, and adrenal OXRs mRNA were determined using semiquantitative reverse transcription-polymerase chain reaction and/or in situ hybridization. Our results indicate that thyroid status affects neither prepro-OX in the hypothalamus nor hypothalamic and adrenal gland OXRs expression.

Expression of growth hormone receptor in the human brain

This study was designed to investigate the presence of growth hormone receptor (GHR) expression in the human brain tissue, both normal and tumoral, as well as in the human glioblastoma cell line U87MG. Reverse transcription-polymerase chain reaction revealed the presence of GHR mRNA in all brain samples investigated and in U87MG cells. GHR immunoreactivity was also detected in this cell line using both immunocytochemistry and western blotting. All together, our data demonstrate the existence of GHR expression within the central nervous system (CNS), thus supporting a possible role for GH in the CNS physiology.

Fas/CD95 Ligation Induces Proliferation of Primary Fetal Astrocytes Through a Mechanism Involving Caspase 8-Mediated ERK Activation

Background: Fas/CD95 is the best-studied member of the death receptor (DR) superfamily in the central nervous system where it can trigger cellular responses other than apoptosis, including the promotion of neurogenesis and neuritogenesis, stimulation of the progression of gliomas, and regulation of the immune response of astrocytes. Methods: We have investigated the role of Fas/CD95 in the regulation of the proliferation of fetal astrocytes in vitro, as well as the signalling pathways involved. Results: Fas/CD95 ligation stimulated the proliferation of primary fetal astrocytes, through a mechanism that depends on the activation of caspase 8 and subsequent phosphorylation of extracellular signal regulated kinase (ERK). Interestingly this proliferative effect is only observed with a low dose of the Fas/CD95 agonist. In contrast, when primary astrocytes are challenged with a high dose of the Fas/CD95 agonist significant cell death occurs. Conclusions: Our findings support that, besides its effects on cell survival, Fas/CD95 may play a complex and prominent role in the regulation of astrocyte proliferation during development.

Bimodal effect of interferon-β on astrocyte proliferation and survival: Importance of nuclear factor-κB

Accumulating evidence indicates that interferon-β (IFN-β) can modify the complex immunopathogenic scenario causing clinical relapse activity and disease progression in MS. However, the beneficial effects of IFN-β in MS patients may also depend on non-immune mechanisms, including the modulation of astrocyte function. In the present report, we have shown that, depending on the dose, IFN-β treatment can either promote astrocyte proliferation and survival, or result astrocyte death. These actions depend, at least in part, on the regulation of nuclear factor-kappa B (NF-κB), an inducible transcription factor present in neurons and glia. This bimodal effect of IFN-β adds a new layer of complexity in the actions of IFN-β within the CNS.

Resistance of neonatal primary astrocytes against Fas-induced apoptosis depends on silencing of caspase 8.

In the present report, we have found that primary fetal astrocytes express caspase 8 and undergo apoptosis in response to Fas ligation. In contrast, neonatal astrocytes do not express detectable levels of the enzyme and are resistant to Fas killing. Fas-induced apoptosis can be restored in these cells by up-regulation of caspase 8 expression by means of transient transfection with a caspase 8-encoding plasmid. Furthermore, treatment of primary astrocytes with the demethylating agent 5-Aza-dC restores caspase 8 expression and increases the sensibility of neonatal astrocytes to the cytotoxic effect of Fas activation. Altogether, our findings indicate that silencing of caspase 8 gene is a key factor controlling the outcome of neonatal astrocytes upon Fas engagement.