Luisa M. Seoane

Influence of Endogenous Leptin Tone on the Estrous Cycle and Luteinizing Hormone Pulsatility in Female Rats

Recent data indicate that leptin may well play an important regulatory role in the hypothalamo-pituitary-gonadal axis. In order to further unravel the mechanisms by which leptin acts, we have studied the effect of treatment (8 days) of leptin antiserum (5 microl daily; i.c.v.) on LH pulsatility and estrous cycle in adult female rats. The administration of leptin antiserum led to a marked decrease in LH pulsatility as assessed by the area under the curve (13.5 +/- 4.7 ng/ml) in comparison to rats treated with normal rabbit serum (114 f 53 ng/ml; p < 0.01). Furthermore, rats treated with leptin antiserum showed an impairment of reproductive function as shown by the fact that all rats remained in anestrus. In conclusion, these data show that leptin markedly influences LH secretion and the estrous cycle in the female rat.

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.

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)

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.

Regulation of in vivo TSH secretion by leptin.

Leptin, the product of the ob gene, is a hormone secreted by adipocytes that regulates food intake and energy expenditure. The hypothalamus-pituitary-thyroid axis is markedly influenced by the metabolic status, being suppressed during food deprivation. The aim of the present study was to assess whether leptin can act as a metabolic signal connecting the adipose tissue with the pituitary-thyroid axis. We studied the effect of leptin administration (10 microg, i.c.v.) on spontaneous TSH secretion and TSH responses to TRH in euthyroid and hypothyroid food-deprived rats. Spontaneous TSH secretion was assessed over 6 h with samples taken every 7 min. Administration of leptin to food-deprived euthyroid rats led to a reversal of the inhibitory effect exerted by fasting on spontaneous TSH secretion. This stimulatory effect of leptin on spontaneous TSH appears to be dependent on the thyroid status since it could not be observed in hypothyroid rats. This data suggests that blunted spontaneous TSH secretion in food-deprived rats is a functional and reversible state, and that the decreased leptin concentrations could be the primary event responsible for the suppression of the hypothalamic-pituitary-thyroid-axis in food-deprived rats.

Influence of thyroid status and growth hormone deficiency on ghrelin

OBJECTIVE To assess whether some of the alterations in energy homeostasis present in thyroid function disorders and GH deficiency could be mediated by ghrelin. DESIGN To assess the influence of thyroid status on ghrelin, adult male Sprague-Dawley rats were treated with vehicle (euthyroid), amino-triazole (hypothyroid) or l-thyroxine (hyperthyroid). The influence of GH on ghrelin was assessed in wild-type (control) and GH-deficient (dwarf) Lewis rats. Evaluation of gastric ghrelin mRNA expression in the stomach was carried out by Northern blot. Circulating levels of ghrelin were measured by radioimmunoassay. RESULTS Hypothyroidism resulted in an increase in gastric ghrelin mRNA levels (euthyroid: 100+/-3.2% vs hypothyroid: 127.3+/-6.5%; P<0.01), being decreased in hyperthyroid rats (70+/-5.4%; P<0.01). In keeping with these results, circulating plasma ghrelin levels were increased in hypothyroid (euthyroid: 124+/-11 pg/ml vs hypothyroid: 262+/-39 pg/ml; P<0.01) and decreased in hyperthyroid rats (75+/-6 pg/ml; P<0.01). Using an experimental model of GH deficiency, namely the dwarf rat, we found a decrease in gastric ghrelin mRNA levels (controls: 100+/-6% vs dwarf: 66+/-5.5%; P<0.01) and circulating plasma ghrelin levels (controls: 124+/-12 pg/ml vs dwarf: 81+/-7 pg/ml; P<0.01). CONCLUSION This study provides the first evidence that ghrelin gene expression is influenced by thyroid hormones and GH status and provides further evidence that ghrelin may play an important role in the alteration of energy homeostasis and body weight present in these pathophysiological states.

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.

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.

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.

Interaction between Leptin and Neuropeptide Y on in vivo Growth Hormone Secretion

Leptin, the product of the ob gene, is a recently discovered hormone secreted by adipocytes that regulates food intake and energy expenditure. Leptin has recently been shown to play a stimulatory role on GH secretion. The aim of the present study was to investigate whether leptin regulation of GH secretion was mediated by hypothalamic neuropeptide Y (NPY). We assessed the effect of leptin administration (10 µg, i.c.v.) and/or NPY (4 µg, i.c.v.) on fasted rats. Furthermore we administered leptin antiserum (10 µl, i.c.v.), anti-NPY serum (5 µl, i.c.v.) or normal rabbit serum (10 µl, i.c.v.) to freely moving fed rats. Spontaneous GH secretion was assessed over 6 h with blood samples taken every 15 min. Fed rats treated with anti-NPY serum exhibited a normal ultradian GH rhythm. However, administration of anti-NPY serum (5 µl, i.c.v., at 120 min) completely reversed the suppression induced by antileptin serum (10 µl, i.c.v., at 0 min) on plasma GH levels (area under the curve, AUC, 168 ± 72 vs. 1,287 ± 430 ng/ml/6 h; p < 0.01). In fasted rats, following NPY administration, GH levels remained suppressed throughout the 6 h studied. Besides, NPY administration completely blunted leptin-induced GH secretion as assessed by the AUC (28.5 ± 11 vs. 520 ± 220 ng/ml/6 h; p < 0.01). Thus, it is possible that NPY mediates the effects of leptin on GH secretion. Alternatively, leptin and NPY could act through parallel pathways to alter GH release with NPY overcoming the stimulatory effect exerted by leptin on plasma GH levels.