María del Carmen Massé García

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)

Role of ghrelin in reproduction.

Ghrelin, the endogenous ligand of GH secretagogue receptor type 1a, has emerged as a pleiotropic modulator of diverse biological functions, including energy homeostasis and, lately reproduction. Here, we review recent reports evaluating the reproductive effects and sites of action of ghrelin, with particular emphasis regarding its role as a molecule integrating reproductive function and energy status. Data gleaned from rodent studies clearly show that besides having direct gonadal effects, ghrelin may participate in the regulation of gonadotropin secretion and it may influence the timing of puberty. In addition, experimental data showing that ghrelin and/or its receptor are expressed in normal human ovary and testis as well as in human ovarian and testicular tumors raise the possibility that the ghrelin system may be involved in the control of cell proliferation in these tumors. We propose that ghrelin either acting as an endocrine and/or paracrine signal may play a major role in the endocrine network that integrates energy balance and reproduction.

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.

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.

Interleukin 6 Deficiency Modulates the Hypothalamic Expression of Energy Balance Regulating Peptides during Pregnancy in Mice

Pregnancy is associated with hyperphagia, increased adiposity and multiple neuroendocrine adaptations. Maternal adipose tissue secretes rising amounts of interleukin 6 (IL6), which acts peripherally modulating metabolic function and centrally increasing energy expenditure and reducing body fat. To explore the role of IL6 in the central mechanisms governing dams energy homeostasis, early, mid and late pregnant (gestational days 7, 13 and 18) wild-type (WT) and Il6 knockout mice (Il6-KO) were compared with virgin controls at diestrus. Food intake, body weight and composition as well as indirect calorimetry measurements were performed in vivo. Anabolic and orexigenic peptides: neuropeptide Y (Npy) and agouti-related peptide (Agrp); and catabolic and anorectic neuropeptides: proopiomelanocortin (Pomc), corticotrophin and thyrotropin-releasing hormone (Crh and Trh) mRNA levels were determined by in situ hybridization. Real time-PCR and western-blot were used for additional tissue gene expression and protein studies. Non-pregnant Il6-KO mice were leaner than WT mice due to a decrease in fat but not in lean body mass. Pregnant Il6-KO mice had higher fat accretion despite similar body weight gain than WT controls. A decreased fat utilization in absence of Il6 might explain this effect, as shown by increased respiratory exchange ratio (RER) in virgin Il6-KO mice. Il6 mRNA levels were markedly enhanced in adipose tissue but reduced in hypothalamus of mid and late pregnant WT mice. Trh expression was also stimulated at gestational day 13 and lack of Il6 blunted this effect. Conversely, in late pregnant mice lessened hypothalamic Il6 receptor alpha (Il6ra), Pomc and Crh mRNA were observed. Il6 deficiency during this stage up-regulated Npy and Agrp expression, while restoring Pomc mRNA levels to virgin values. Together these results demonstrate that IL6/IL6Ra system modulates Npy/Agrp, Pomc and Trh expression during mouse pregnancy, supporting a role of IL6 in the central regulation of body fat in this physiological state.

Divergent responses to thermogenic stimuli in BAT and subcutaneous adipose tissue from interleukin 18 and interleukin 18 receptor 1-deficient mice.

Brown and beige adipocytes recruitment in brown (BAT) or white adipose tissue, mainly in the inguinal fat pad (iWAT), meet the need for temperature adaptation in cold-exposure conditions and protect against obesity in face of hypercaloric diets. Using interleukin18 (Il18) and Il18 receptor 1- knockout (Il18r1-KO) mice, this study aimed to investigate the role of IL18 signaling in BAT and iWAT activation and thermogenesis under both stimuli. Il18-KO, extremely dietary obesity-prone as previously described, failed to develop diet-induced thermogenesis as assessed by BAT and iWAT Ucp1 mRNA levels. Overweight when fed standard chow but not HFD, HFD-fed Il18r1-KO mice exhibited increased iWAT Ucp1 gene expression. Energy expenditure was reduced in pre-obese Il18r1-KO mice and restored upon HFD-challenge. Cold exposure lead to similar results; Il18r1-KO mice were protected against acute body temperature drop, displaying a more brown-like structure, alternative macrophage activation and thermogenic gene expression in iWAT than WT controls. Opposite effects were observed in Il18-KO mice. Thus, Il18 and Il18r1 genetic ablation disparate effects on energy homeostasis are likely mediated by divergent BAT responses to thermogenic stimuli as well as iWAT browning. These results suggest that a more complex receptor-signaling system mediates the IL18 adipose-tissue specific effects in energy expenditure.

Regulation of Body Weight Homeostasis During Pregnancy and Lactation

Pregnancy is a hypermetabolic state in which a great increase in maternal body fat and weight occurs, mostly in the final trimester of gestation, and it is associated with relevant neuroendocrine changes as adaptations to the new hormonal status. Data gleaned over the last few years have allowed the characterization of different central and peripheral signals involved in the regulation of body weight homeostasis.