Clara V. Alvarez

Hypothalamic Fatty Acid Metabolism Mediates the Orexigenic Action of Ghrelin

Current evidence suggests that hypothalamic fatty acid metabolism may play a role in regulating food intake; however, confirmation that it is a physiologically relevant regulatory system of feeding is still incomplete. Here, we use pharmacological and genetic approaches to demonstrate that the physiological orexigenic response to ghrelin involves specific inhibition of fatty acid biosynthesis induced by AMP-activated protein kinase (AMPK) resulting in decreased hypothalamic levels of malonyl-CoA and increased carnitine palmitoyltransferase 1 (CPT1) activity. In addition, we also demonstrate that fasting downregulates fatty acid synthase (FAS) in a region-specific manner and that this effect is mediated by an AMPK and ghrelin-dependent mechanisms. Thus, decreasing AMPK activity in the ventromedial nucleus of the hypothalamus (VMH) is sufficient to inhibit ghrelins effects on FAS expression and feeding. Overall, our results indicate that modulation of hypothalamic fatty acid metabolism specifically in the VMH in response to ghrelin is a physiological mechanism that controls feeding.

A GRFa2/Prop1/Stem (GPS) Cell Niche in the Pituitary

Background The adult endocrine pituitary is known to host several hormone-producing cells regulating major physiological processes during life. Some candidates to progenitor/stem cells have been proposed. However, not much is known about pituitary cell renewal throughout life and its homeostatic regulation during specific physiological changes, such as puberty or pregnancy, or in pathological conditions such as tumor development. Principal Findings We have identified in rodents and humans a niche of non-endocrine cells characterized by the expression of GFRa2, a Ret co-receptor for Neurturin. These cells also express b-Catenin and E-cadherin in an oriented manner suggesting a planar polarity organization for the niche. In addition, cells in the niche uniquely express the pituitary-specific transcription factor Prop1, as well as known progenitor/stem markers such as Sox2, Sox9 and Oct4. Half of these GPS (GFRa2/Prop1/Stem) cells express S-100 whereas surrounding elongated cells in contact with GPS cells express Vimentin. GFRa2+-cells form non-endocrine spheroids in culture. These spheroids can be differentiated to hormone-producing cells or neurons outlining the neuroectoderm potential of these progenitors. In vivo, GPSs cells display slow proliferation after birth, retain BrdU label and show long telomeres in its nuclei, indicating progenitor/stem cell properties in vivo. Significance Our results suggest the presence in the adult pituitary of a specific niche of cells characterized by the expression of GFRa2, the pituitary-specific protein Prop1 and stem cell markers. These GPS cells are able to produce different hormone-producing and neuron-like cells and they may therefore contribute to postnatal pituitary homeostasis. Indeed, the relative abundance of GPS numbers is altered in Cdk4-deficient mice, a model of hypopituitarism induced by the lack of this cyclin-dependent kinase. Thus, GPS cells may display functional relevance in the physiological expansion of the pituitary gland throughout life as well as protection from pituitary disease.

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.

The dependence receptor Ret induces apoptosis in somatotrophs through a Pit-1/p53 pathway, preventing tumor growth

Somatotrophs are the only pituitary cells that express Ret, GFRα1 and GDNF. This study investigated the effects of Ret in a somatotroph cell line, in primary pituitary cultures and in Ret KO mice. Ret regulates somatotroph numbers by inducing Pit‐1 overexpression, leading to increased p53 expression and apoptosis, both of which can be prevented with Ret or Pit‐1 siRNA. The Pit‐1 overexpression is mediated by sustained activation of PKCδ, JNK, c/EBPα and CREB induced by a complex of Ret, caspase 3 and PKCδ. In the presence of GDNF, Akt is activated, and the Pit‐1 overexpression and resulting apoptosis are blocked. The adenopituitary of Ret KO mice is larger than normal, showing Pit‐1 and somatotroph hyperplasia. In normal animals, activation of the Ret/Pit‐1/p53 pathway by retroviral introduction of Ret blocked tumor growth in vivo. Thus, somatotrophs have an intrinsic mechanism for controlling Pit‐1/GH production through an apoptotic/survival pathway. Ret might be of value for treatment of pituitary adenomas.

Evidence for a Direct Pituitary Inhibition by Free Fatty Acids of in vivo Growth Hormone Responses to Growth Hormone-Releasing Hormone in the Rat

The aim of this study was to determinate whether elevations in circulating free fatty acids (FFA) inhibit in vivo growth hormone (GH) responses to GH-releasing hormone (GHRH) by increasing hypothalamic somatostatin release or by acting directly on the pituitary. Thus, we have studied the effect of an Intralipid-heparin infusion on in vivo GH responses to GHRH in normal rats, normal rats passively immunized with antisomatostatin antiserum, rats with medial hypothalamic ablation, and hypophysectomized rats bearing two hypophyses under the renal capsule. Administration of 1 ml of Intralipid (500 microliters at -30 min and 500 microliters at -25 min) plus heparin (50 IU at -15 min) induced a marked decrease in GH responses to both 1 and 5 micrograms/kg of GHRH (p less than 0.01 at 5, 10 and 15 min for GHRH alone vs. GHRH plus Intralipid). A similar degree of inhibition was obtained after the administration of antisomatostatin antiserum (750 microliters i.v. at -60 min) previous to a challenge with 5 micrograms/kg of GHRH plus 1 ml of Intralipid (p less than 0.05 at 5 and 15 min, and p less than 0.01 at 10 min for GHRH plus normal rabbit serum vs. GHRH plus Intralipid plus antisomatostatin antiserum). Furthermore, administration of 1 ml of Intralipid also markedly reduced GH responses to GHRH in rats with medial hypothalamic ablation (p less than 0.01 at 5, 10, 15 and 30 min for GHRH alone vs. GHRH plus Intralipid) as well as in hypophysectomized rats bearing two hypophyses under the renal capsule (p less than 0.01 at 5, 10 and 15 min for GHRH alone vs. GHRH plus Intralipid).(ABSTRACT TRUNCATED AT 250 WORDS)

TGF-β1 actions on FRTL-5 cells provide a model for the physiological regulation of thyroid growth

Little is known about the TGF-β1 mechanism that promotes thyroid cell growth arrest. We assessed TGF-β1 effects on Fisher rat thyroid cell line (FRTL-5). This allowed us to study TGF-β1 action on thyroid cells in various physiological situations such as actively proliferating cells, resting cells stimulated to proliferate by the action of various mitogens, and resting cells. TGF-β1 arrested proliferating FRTL-5 cells, increasing c-myc mRNA levels and reducing p27-free cyclin D1 protein levels, without affecting either the cellular content of p27 or the cyclin D1-p27 complexes. Moreover, TGF-β1 treatment reduced the activity of cyclin E-CDK2 complexes and, consequently, pRB was found to be hypophosphorylated. TGF-β1 prevented resting cells to enter in the cell cycle when stimulated with growing medium (newborn calf serum plus a mixture of five hormones) but not when TSH (thyroid stimulating hormone) plus IGF-1 (Insulin-like growth factor I) were used as mitogens. Both stimuli increased the levels of cyclins D1, D3 and E but TGF-β1 had a greater effect in decreasing these cyclin levels in growing-medium stimulated cells than in TSH+IGF-1. This suggests that for FRTL-5 cells, the content of these cyclins must exceed a threshold to progress through the cell cycle. TGF-β1 induced apoptosis in quiescent cells, accompanied by a reduction in p27 protein levels and an increase in c-myc expression. Interestingly, TGF-β1-induced variations in prothymosin alpha and c-myc mRNA levels were not correlated. TGF-β1 always promoted an increase of p15 mRNA levels. In summary, our results point to the fact that TGF-β1 could play a physiological role in the control of thyroid growth through the modification of cell cycle regulatory proteins.

Regulation of His-dTrp-Ala-Trp-dPhe-Lys-NH2 (GHRP-6)-lnduced GH Secretion in the Rat

His-dTrp-Ala-Trp-dPhe,Lys-NH2(GHRP-6) is a synthetic compound that releases GH in a dose-response and specific manner in several species and that may well be related to an endogenous compound of similar structure. The aim of this study was to investigate the in vivo GH responses to GHRP-6 in pentobarbital anesthetized rats. Specifically and in order to avoid the influence of endogenous GHRH and somatostatin secretion we studied the GH responses to GHRP-6 in animals with surgical ablation of the hypothalamus, confirmed by histological assessment, as well as in hypophysectomyzed-transplanted rats bearing two hypophyses under the renal capsule. Since it has been previously reported that rats pretreated with GHRH (10 micrograms/kg i.p. every 12 h for 15 days) rather than saline-treated rats have greater GH responses to acutely administered GHRH, we compared the self-potentiating effect of chronic GH pretreatment with GHRP-6 (10 micrograms/kg i.p. every 12 h). Furthermore we also studied the influence of estrogens, glucocorticoids, free fatty acids (FFA) and bombesin on somatotroph responsiveness to GHRP-6 in intact rats. We found a greater GH response to GHRP-6 in rats that underwent a surgical ablation of the hypothalamus 36 h prior to the test than in sham-operated rats. A direct stimulatory effect of GHRP-6 on in vivo GH secretion was demonstrated by a clear GH response to GHRP-6 in hypophysectomyzed-transplanted rats. In addition, we found a similar response whether the animals were pretreated with GHRH or GHRP-6 over the previous 2 weeks. Finally, we found that both estrogen- and testosterone-treated rats have greater GH responses to GHRP-6 than untreated rats. On the other hand, chronic dexamethasone administration, acute elevation of circulating FFA levels and bombesin administration markedly inhibited GH responses to GHRP-6. In contrast to the effects exerted on GH responses to GHRP-6 estrogen administration led to a decrease in GH responses to GHRH while dexamethasone did not affect the GH responses to GHRH, highlighting a differential regulation of these hormones on somatotroph responsiveness to these peptides.

Craniopharyngiomas Express Embryonic Stem Cell Markers (SOX2, OCT4, KLF4, and SOX9) as Pituitary Stem Cells but Do Not Coexpress RET/GFRA3 Receptors

CONTEXT Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). OBJECTIVE Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathkes pouch remnants, express similar markers as normal pituitary stem cells. DESIGN We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). RESULTS Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. CONCLUSIONS Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated growth of CRF.

Deletion of miRNA processing enzyme Dicer in POMC-expressing cells leads to pituitary dysfunction, neurodegeneration and development of obesity.

MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. However, their potential role in the central regulation of whole-body energy homeostasis is still unknown. In this study we show that the expression of Dicer, an essential endoribonuclease for miRNA maturation, is modulated by nutrient availability and excess in the hypothalamus. Conditional deletion of Dicer in POMC-expressing cells resulted in obesity, characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism and alterations in the pituitary-adrenal axis. The development of the obese phenotype was paralleled by a POMC neuron degenerative process that started around 3 weeks of age. Hypothalamic transcriptomic analysis in presymptomatic POMCDicerKO mice revealed the downregulation of genes implicated in biological pathways associated with classical neurodegenerative disorders, such as MAPK signaling, ubiquitin-proteosome system, autophagy and ribosome biosynthesis. Collectively, our results highlight a key role for miRNAs in POMC neuron survival and the consequent development of neurodegenerative obesity.

TGF-β-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-κB activation

Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis. TGF-β is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-β in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Graves disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-β induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-β induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apoptosis in the absence of TGF-β. By contrast, in cells in which p27 was overexpressed, TGF-β had a survival effect. In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-κB survival program. Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-β induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-κB program.