Lipoprotein internalisation induced by oncogenic AMPK activation is essential to maintain glioblastoma cell growth.

AIM OF THE STUDY: Metabolic adaptations are essential during tumour growth to maintain the high proliferation levels exhibited by cancer cells. In this study, we examined the transformations that occurred in the lipid metabolism in astrocytic tumours, and the possible role of the fuel-sensing enzyme AMPK. Metabolic targets might help design new and effective drugs for cancer. METHODS: To accomplish this objective, we studied both mice and human astrocytic tumours. We first used a mouse model of astrocytoma driven by oncogenic H-RasV12 and/or with PTEN deletion based on the common constitutive activation of the Raf/MEK/ERK and PI3K/AKT cascades in human astrocytomas. We then confirmed the results in human glioblastoma cell lines and in glioblastoma tissue samples from patients. RESULTS: We show that the high levels of activated AMPK, observed in astrocytic tumours, increase extracellular lipid internalisation and reduce energy expenditure by inhibiting 'de novo' fatty acid (FA) synthesis, which allows tumour cells to obtain building blocks and energy to be able to create new organelles and new cells. CONCLUSIONS: Our findings demonstrate that AMPK plays a crucial role in glioblastoma cell growth and suggest that blocking lipoprotein receptors could potentially be used as a plausible therapeutic approach for these and other type of tumours with high levels of AMPK.

Interleukin-6 deletion in mice driven by aP2-Cre-ERT2 prevents against high-fat diet-induced gain weight and adiposity in female mice.

AIM: Interleukin-6 (IL-6) is a major cytokine controlling body weight and metabolism, but because many types of cells can synthesize and respond to IL-6 considerable uncertainty still exists about the mechanisms underlying IL-6 effects. Therefore, the aim of this study was to analyse the effects of tissue-specific deletion of IL-6 using a fatty acid binding protein (aP2) promoter-Cre inducible system (aP2-Cre-ERT2). METHODS: Tissue-specific IL-6 KO mice (aP2-IL-6 KO mice) were produced upon tamoxifen administration and were fed a high-fat diet (HFD, 58.4% kcal from fat) or a control diet (18%) for 14 weeks. RESULTS: aP2-IL-6 KO female mice on a HFD gained less weight and adiposity than littermate wild-type mice, but these effects were not observed in males. Hypothalamic factors such as NPY and AgRP showed a pattern of expression consistent with this sex-specific phenotype. PGC-1α expression was increased in several tissues in aP2-IL-6 KO female mice, which is compatible with increased energy expenditure. Serum leptin, insulin, glucose, cholesterol and triglycerides levels were increased by HFD, and in females IL-6 deficiency reversed this effect in the case of insulin and cholesterol. HFD induced impaired responses to insulin and glucose tolerance tests, but no significant differences between genotypes were observed. CONCLUSION: The present results demonstrate that deletion of IL-6 driven by aP2-Cre regulates body weight, body fat and metabolism in a sex-specific fashion.

Interleukin-6 regulates the expression of hypothalamic neuropeptides involved in body weight in a gender-dependent way.

Interleukin (IL)-6 has been involved in the control of body weight and body fat. Nevertheless, the mechanisms underlying these effects are not completely understood because central and peripheral actions of IL-6 are plausible. To gain further insight into the central effects of IL-6, we used transgenic mice expressing the IL-6 gene under the control of the glial fibrillary acidic protein (GFAP) promoter (GFAP-IL-6 mice), therefore with central nervous system-restricted over-expression of IL-6, and we studied the expression of the main neuropeptides responsible for energy homeostasis in specific hypothalamic nuclei. Neuropeptide Y (NPY), agouti-related peptide (AgRP), melanin-concentrating hormone (MCH), prepro-orexin (preproOX) (orexigenic and anabolic neuropeptides), pro-opiomelanocortin (POMC) and corticotrophin-releasing hormone (CRH) (anorexigenic and catabolic peptides) mRNA levels were determined using in situ hybridisation in young (2-4 month-old) and old (10-12 month-old) female and male mice under different feeding conditions: normal diet (control) and high-fat diet (HFD), and 24 h-food deprivation. In GFAP-IL-6 females fed a control diet (GFAP-IL-6-control), we showed a significant decrease in NPY and AgRP mRNA levels at all ages, and a late increase in POMC expression (only significant in older animals). These differences were blunted in HFD mice. By contrast, GFAP-IL-6-control males showed a decrease in CRH mRNA content at early ages (2-4 months), and an increase in older mice (10-12 months). Interestingly, these differences were again blunted in HFD mice. Finally, central IL-6 was not able to counteract the effects of 24 h of fasting on body weight, plasma glucose levels and the mRNA content of the peptides evaluated in the present study. Our results demonstrate that IL-6 may regulate the expression of hypothalamic neuropeptides involved in the control of body weight and body fat acting at the central level in a gender- and age-dependent way.

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 ß-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.

The vagus nerve as a regulator of growth hormone secretion.

The growth hormone (GH) axis is mainly regulated by the growth hormone releasing hormone (GHRH) and somatostatin (SS) hypothalamic peptides. Nevertheless, since ghrelin peptide was discovered as the natural ligand for growth hormone secretagogue receptor (GHS-R), the mechanism of GH regulation has acquired a new dimension. It was described that ghrelin possesses a relevant effect inducing GH secretion when it is administered peripherally. A role of the vagus nerve mediating ghrelin action has been described although this effect is not understood. Under this context the main objective of this work was to determine the possible involvement of the vagus in the somatotroph axis regulation. The results in this manuscript show that animals with a disruption of the vagus connection presented lower basal IGF-1 and GH levels, a decreased GH response to peripheral GHRH administration and a marked diminution in the GH response to peripheral and central ghrelin treatments. In addition, vagotomized animals showed a down-regulation of GHRH mRNA in the arcuate nucleus of the hypothalamus and a down-regulation in both GHRH and GHS receptors' mRNA at the pituitary level. In conclusion, the present work reveals that the vagus nerve is crucial in growth hormone regulation and essential for the full GH-releasing effect of ghrelin.

Peripheral endocannabinoid system-mediated actions of rimonabant on growth hormone secretion are ghrelin-dependent.

The somatotroph axis is a crucial pathway regulating metabolism. Despite the fact that the endocannabinoid system has been also revealed as a potent modulator of energy homeostasis, little information is available concerning a putative interaction between these two systems. The aim of the present study was to determine the in vivo effects of the blockade of the cannabinoid receptor type 1 (CB1) over growth hormone (GH) secretion using the CB1 antagonist rimonabant. The results obtained show that the blockade of the CB1 peripheral receptor by i.p. injection of rimonabant significantly inhibited pulsatile GH secretion. Similarly, it was found that this injection significantly decreased ghrelin-induced GH secretion without any effect on growth hormone-releasing hormone (GHRH)-induced GH discharge. In situ hybridisation showed that the peripheral blockade of CB1 did not affect hypothalamic somatostatin mRNA levels; however, GHRH mRNA expression was significantly decreased. The blockade of the vagus nerve signal by surgical vagotomy eliminated the inhibitory action of rimonabant on GHRH mRNA and consequently on GH. On the other hand, the central CB1 blockade by i.c.v. rimonabant treatment was unable to reproduce the effect of peripheral blockade on GHRH mRNA, nor the GH response to ghrelin. In conclusion, the data reported in the present study establish, from a physiological point of view, the existence of a novel mechanism of GH regulation implicating the action of the cannabinoid receptor on the somatotroph axis.

Perinatal overfeeding in rats results in increased levels of plasma leptin but unchanged cerebrospinal leptin in adulthood.

OBJECTIVE: To study the effect of perinatal programming and overfeeding on the hypothalamic control mechanisms of food intake in adult rats. DESIGN: Neonatal programming effects on body weight, food intake, central and peripheral leptin levels, hypothalamic neuropeptides, leptin receptors and central leptin responsiveness in adult rats. MEASUREMENTS: Plasma and cerebrospinal fluid (CSF) leptin levels were analyzed using radioimmunoassay. Neuropeptide mRNA levels were analyzed using in situ hybridization. Leptin receptor mRNA levels were analyzed using reverse transcriptase-polymerase chain reaction. RESULTS: Perinatally overfed rats growing up in small litters (SL) maintain their obese and hyperleptinemic phenotype in adulthood. However, leptin levels in CSF are abnormally low considering the plasmatic hyperleptinemia. In contrast to the already reported changes in perinatally overfed juvenile rats, perinatally overfed adult rats did not show any alteration in the expression of leptin receptor isoforms and evaluated neuropeptides. Moreover, SL adult rats showed a normal sensitivity regarding the inhibitory effect of intracerebroventricular leptin administration on food intake. CONCLUSION: Perinatal overfeeding does not induce alterations in either the anorectic response to central leptin administration or expression of leptin receptors and neuropeptides in adulthood. The leptin resistance to peripheral leptin in SL adult rats may be related to impaired leptin transport across the blood-brain barrier.

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

AIM/HYPOTHESIS: Perinatal 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. METHODS: Leptin levels were analysed using RIA. Leptin receptor mRNA levels were analysed using RT-PCR. Neuropeptide mRNA levels were analysed using in situ hybridisation. RESULTS: Perinatally 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/INTERPRETATION: Rats 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.

Orexin A suppresses in vivo GH secretion.

BACKGROUND/AIMS: Orexins (OXs) are a newly described family of hypothalamic neuropeptides. Based on the distribution of OX neurons and their receptors in the brain, it has been postulated that they could play a role in the regulation of neuroendocrine function. GH secretion is markedly influenced by nutritional status and body weight. To investigate the role OX-A plays in the neuroregulation of GH secretion we have studied its effect on spontaneous GH secretion as well as GH responses to GHRH and ghrelin in freely moving rats. Finally, we also assessed the effect of OX-A on in vitro GH secretion. METHODS: We administered OX-A (10 microg, i.c.v.) or vehicle (10 microl, i.c.v.) to freely moving rats. Spontaneous GH secretion was assessed over 6 h with blood samples taken every 15 min. RESULTS: Administration of OX-A led to a decrease in spontaneous GH secretion in comparison with vehicle-treated rats, as assessed by mean GH levels (means+/-s.e.m. 4.2+/-1.7 ng/ml vs 9.4+/-2.2 ng/ml; P<0.05), mean GH amplitude (3.6+/-0.5 ng/ml vs 20.8+/-5.6 ng/ml; P<0.01) and area under the curve (848+/-379 ng/ml per 4 h vs 1957+/-458 ng/ml per 4 h; P<0.05). In contrast, OX-A failed to modify in vivo GH responses to GHRH (10 microg/kg, i.v.) although it markedly blunted GH responses to ghrelin (40 microg/kg, i.v.) (mean peak GH levels: 331+/-71 ng/ml, vehicle, vs 43+/-11 ng/ml in OX-A-treated rats; P<0.01). Finally, OX-A infusion (10(-7), 10(-8) or 10(-9) M) failed to modify in vitro basal GH secretion or GH responses to GHRH, ghrelin and KCl. CONCLUSIONS: These data indicate that OX-A plays an inhibitory role in GH secretion and may act as a bridge among the regulatory signals that are involved in the control of growth, nutritional status and sleep regulation.

Orexin 1 receptor messenger ribonucleic acid expression and stimulation of testosterone secretion by orexin-A in rat testis.

Orexins are hypothalamic neuropeptides primarily involved in the regulation of food intake and arousal states. In addition, a role for orexins as central neuroendocrine modulators of reproductive function has recently emerged. Prepro-orexin and orexin type-1 receptor mRNAs have been detected in the rat testis. This raises the possibility of additional peripheral actions of orexins in the control of reproductive axis, which remains so far unexplored. To analyze the biological effects and mechanisms of action of orexins in the male gonad, we evaluated testicular expression of orexin receptor 1 (OX(1)R) and orexin receptor 2 (OX(2)R) mRNAs in different experimental settings and the effect of orexin-A on testicular testosterone (T) secretion. Persistent expression of OX(1)R mRNA was demonstrated in the rat testis throughout postnatal development. In contrast, OX(2)R transcript was not detected at any developmental stage. Expression of OX(1)R mRNA persisted after selective elimination of mature Leydig cells and was detected in isolated seminiferous tubules at defined stages of the seminiferous epithelial cycle. In addition, testicular OX(1)R mRNA expression appeared to be under hormonal regulation; it was reduced by long-term hypophysectomy and partially restored by FSH replacement, whereas down-regulation was observed after exposure to increasing doses of the ligand in vitro. Moreover, OX(1)R mRNA expression was sensitive to neonatal imprinting by estrogen. Finally, orexin-A, in a dose-dependent manner, significantly increased basal, but not human choriogonadotropin-stimulated, T secretion in vitro. A similar stimulatory effect was observed in vivo after intratesticular administration of orexin-A. In conclusion, our present results provide the first evidence for the regulated expression of OX(1)R mRNA and functional role of orexin-A in the rat testis. Overall, our data are suggestive of a novel site of action of orexins in the control of male reproductive axis.

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

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.

Cellular localization of orexin receptors in human pituitary.

Orexins-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and relationated with the stimulation of food intake. They act on G protein receptors named orexin receptor 1 (OX(1)R) and orexin receptor 2 (OX(2)R), respectively. In the present study, we used RT-PCR and immunohistochemical techniques to detect the presence of OX(1)R and OX(2)R in human pituitary. A band of the expected size for both OX(1)R and OX(2)R was shown in human pituitary by RT-PCR. The cellular localization of OX(1)R and OX(2)R was carried out using histological techniques. By consecutive sections we demonstrated that OX(1)R was present in acidophil, diffusely distributed cells, which represent the half of the total adenohypophysis cell population. As was expected, these cells were shown to coexpress GH. OX(2)R was found in the pars intermedia and in clusters of basophil cells of the anterior pituitary, which coexpress ACTH. These results were confirmed by double immunofluorescence techniques. We also found focal positivity in axon terminals of neurohypophysis, more intense for OX(2)R than for OX(1)R. In conclusion, these results demonstrated for the first time that OX(1)R and OX(2)R were expressed by somatotrope and corticotrope cells, respectively.

Cellular localization of orexin receptors in human pituitary.

Orexins-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and relationated with the stimulation of food intake. They act on G protein receptors named orexin receptor 1 (OX(1)R) and orexin receptor 2 (OX(2)R), respectively. In the present study, we used RT-PCR and immunohistochemical techniques to detect the presence of OX(1)R and OX(2)R in human pituitary. A band of the expected size for both OX(1)R and OX(2)R was shown in human pituitary by RT-PCR. The cellular localization of OX(1)R and OX(2)R was carried out using histological techniques. By consecutive sections we demonstrated that OX(1)R was present in acidophil, diffusely distributed cells, which represent the half of the total adenohypophysis cell population. As was expected, these cells were shown to coexpress GH. OX(2)R was found in the pars intermedia and in clusters of basophil cells of the anterior pituitary, which coexpress ACTH. These results were confirmed by double immunofluorescence techniques. We also found focal positivity in axon terminals of neurohypophysis, more intense for OX(2)R than for OX(1)R. In conclusion, these results demonstrated for the first time that OX(1)R and OX(2)R were expressed by somatotrope and corticotrope cells, respectively.

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.

Regulation of somatotroph cell function by the adipose tissue.

Obese subjects exhibit a marked decrease in plasma growth hormone (GH) levels. However, the mechanisms by which increased adiposity leads to an impairment of GH secretion are poorly understood. Recent evidence suggests that the adipose tissue can markedly influence GH secretion via two different signals, namely free fatty acids (FFA) and leptin. FFA appear to inhibit GH secretion mainly by acting directly at pituitary level. Interestingly, reduction in circulating FFA levels in obese subjects led to a marked increase in GH responses to different GH secretagogues. This indicates that FFA exert a tonic inhibitory effect that contributes to blunted GH secretion in obese subjects. Recent data have shown that leptin is a metabolic signal that regulates GH secretion, since the administration of leptin antiserum to adult rats led to a marked decrease in spontaneous GH secretion. However, leptin prevents,the inhibitory effect exerted by fasting on plasma GH levels. The effect of leptin in adult rats appears to be exerted at hypothalamic level by regulating growth hormone releasing hormone (GHRH), somatostatin and neuropeptide Y (NPY)-producing neurones. In addition, during fetal life or following the development of pituitary tumors, leptin can also act directly at the anterior pituitary.

Leptin regulation of prepro-orexin and orexin receptor mRNA levels in the hypothalamus.

The aim of this study was to determine the effects of leptin treatment on prepro-orexin and orexin receptor expression in the rat hypothalamus. Adult male rats, food-deprived for 48 and 72 h, were treated one time with vehicle or leptin (10 microg, icv). Prepro-orexin mRNA content was measured by semiquantitative RT-PCR, Northern blot, and in situ hybridization; orexin receptor 1 and 2 mRNA content was quantified by Northern blot and/or semiquantitative RT-PCR. Our results indicate that leptin inhibits a fasting-induced increase in prepro-orexin mRNA and orexin receptor 1 mRNA levels in the rat hypothalamus, while orexin receptor 2 mRNA levels were unchanged in all situations evaluated. These data provide direct evidence for an additional mechanism of adaptation of the hypothalamus to food deprivation and for a new effect of leptin in the regulation of food intake.

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.

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.

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.

Leptin increases in vivo GH responses to GHRH and GH-releasing peptide-6 in food-deprived rats.

BACKGROUND: Leptin has recently been shown to have a stimulatory effect on basal GH secretion. However, the mechanisms by which leptin exert this effect are not yet clear. GHRH and GH-releasing peptide (GHRP)-6 are the two most potent GH secretagogues described to date. OBJECTIVE: To determine if leptin could also enhance in vivo GH responses to a maximal dose of GHRH. DESIGN: Leptin (10microg i.c.v.) or vehicle was administered at random before GHRH (10microg/kg i,v.) or GHRP-6 (50microg/kg i.v.), to freely-moving rats with food available ad libitum and to (48h) food-deprived rats. METHODS: Leptin and GH concentrations were measured by radioimmunoassay. Comparison between the different groups was assessed by the Mann-Whitney test. RESULTS: In comparison with fed rats, food-deprived rats showed a marked decrease in GH responses to GHRH as assessed by the area under the curve (5492+/-190ng/ml in fed rats and 1940+/-128ng/ml in fasted rats; P<0.05) and GHRP-6 (3695+/-450 in fed rats and 1432+/-229 in fasted rats; P<0.05). In comparison with its effects in vehicle-treated rats, leptin administered to food-deprived rats markedly increased GH responses to both GHRH (6625+/-613ng/ml; P<0.05) and GHRP-6 (5862+/-441ng/ml; P<0.05). CONCLUSIONS: These data suggest that the blunted GH response to GHRH and GHRP-6 in food-deprived rats is a functional and reversible state, and that the decreased leptin concentrations could be the primary defect responsible for the altered GH secretion in food-deprived rats.

Role of growth hormone receptor in HL-60 cell survival.

Although it is presently well established that locally produced growth hormone (GH) plays a major role in the regulation of survival mechanisms in hemopoietic cells, the responsible mechanisms are poorly understood, and the involvement of the GH receptor (GHR) has not even been demonstrated to date. In this work we investigated the presence of GHR in the human promyelocytic leukemia cell line HL-60, as well as the ability of GH treatment to stimulate both GHR and survival signaling pathways downstream GHR. Our results demonstrate that (1) both GHR mRNA and GHR immunoreactivity are present in HL-60 cells; (2) GH treatment results in an increase in the phosphorylation of the GHR-associated Jak2 and Stat3 proteins, indicating the ability of the hormone to induce receptor activation; and (3) activation of GHR increases the activity of Akt, a serine/threonine kinase that plays a prominent role in the regulation of cell survival. Taken together, these results demonstrate that GHR activation promotes survival of HL-60 cells, thus suggesting that GH plays a major role in the regulation of cell survival in the hemopoietic system, via an autocrine/paracrine mechanism.