Hypothalamic Releasing Hormone Research Articles

Metabolic and hormonal control of the desire for food and sex: Implications for obesity and eating disorders

During evolution, the ability to overeat and store the extra energy as glycogen and lipids in specialized tissues must have conferred a reproductive advantage by releasing animals from the need to eat constantly, enabling them to engage in behaviors that improved reproductive success. Mechanisms that inhibited ingestive behavior might have been most adaptive when they caused individuals to stop foraging, hoarding and eating in order to find and court potential mates. Conversely, the ability to abstain from reproductive activities to engage in foraging and eating was probably critical for individual survival during severe energetic challenges because reproductive processes are energetically costly and can be delayed until the energetic conditions improve. The mechanisms that control ingestive behavior most likely evolved under conditions in which both food and mates were available, and thus, our understanding might be limited by our narrow focus on food intake in animals isolated from potential mates, and reproductive behaviors in the absence of food. Our understanding of obesity and eating disorders will be enriched by the study of the choice between ingestive and reproductive behaviors and by a renewed attention to “reproductive” hormones such as gonadal steroids and hypothalamic releasing hormones. Furthermore, leptin and reproductive hormones have both organizational and activational effects on the energy balancing system including those mechanisms that control appetite, body fat content and body fat distribution. Understanding these organizational and activational effects on body fat distribution might lead to a better understanding of sex differences in the propensity to develop obesity, type II diabetes and eating disorders.

Changes in Expression of Hypothalamic Releasing Hormone Receptors in Individual Rat Anterior Pituitary Cells during Maturation, Puberty and Senescence

Anterior pituitary (AP) is formed by five different cell types, each one producing a different AP hormone whose secretion is regulated by a specific hypothalamic-releasing hormone (HRH). On the other hand, a significant number of AP cells express multiple HRH receptors (multiresponsive cells). Plastic changes in expression of HRH receptors in individual AP cells are involved in critical endocrine events. Here we have characterized the changes in functional responses to CRH, LHRH, TRH, and GHRH in individual AP cells throughout the whole life span of the rat. To this end, calcium responses to the HRHs were followed by single-cell imaging in freshly dispersed AP cells prepared from rats of different ages (0-540 postnatal days). Three different cell pools were identified: 1) monoresponsive cells, holding a single class of HRH receptor; 2) multiresponsive cells; and 3) nonresponsive cells. The relative abundance of each pool changed with age. Nonresponsive cells were abundant at birth, multiresponsive cells were abundant at puberty, and monoresponsive cells dominated at senescence. The relative abundance of each HRH receptor changed largely with age but not gender. In addition, the contribution of monoresponsive and multiresponsive cells to responses to each HRH changed very much with age. Thus, the anterior pituitary shows large changes in cell populations typed by functional responses to HRHs during maturation, puberty, and senescence.

A germ line mutation that delays prostate cancer progression and prolongs survival in a murine prostate cancer model

Circulating insulin-like growth factor-I (IGF-I) levels have been shown to be related to risk of prostate cancer in epidemiologic studies. While specific genetic loci responsible for interindividual variation in circulating IGF-I levels in normal men have not been identified, candidate genes include those involved in the growth hormone (GH)-IGF-I axis such as the hypothalamic factors GH releasing hormone (GHRH) and somatostatin and their receptors. To investigate the role of the GH-IGF-I axis on in vivo prostate carcinogenesis and neoplastic progression, we generated mice genetically predisposed to prostate cancer (the TRAMP model) to be homozygous for lit, a mutation that inactivates the GHRH receptor (GHRH-R) and reduces circulating levels of GH and IGF-I. The lit mutation significantly reduced the percentage of the prostate gland showing neoplastic changes at 35 weeks of age (P=0.0005) and was also associated with improved survival (P<0.01). These data provide an example of a germ line mutation that reduces risk in an experimental prostate carcinogenesis model. The results suggest that prostate carcinogenesis and progression may be influenced by germ line variation of genes encoding signalling molecules in the GH-IGF-I axis.

Physiology of ghrelin and related peptides

Growth hormone (GH) released from pituitary under direct control of hypothalamic releasing (i.e., GHRH) and inhibiting (i.e., sst or SRIF) hormones is an anabolic hormone that regulates metabolism of proteins, fats, sugars and minerals in mammals. Cyril Bowers’ discovery of GH-releasing peptide (GHRP-6) was followed by a search for synthetic peptide and nonpeptide GH-secretagogues (GHSs) that stimulate GH release, as well as a receptor(s) unique from GHRH receptor. GHRH and GHSs operate through distinct G protein-coupled receptors to release GH. Signal transduction pathways activated by GHS increase intracellular Ca 2+ concentration in somatotrophs, whereas GHRH increases cAMP. Isolation and characterization of ghrelin, the natural ligand for GHS receptor, has opened a new era of understanding to physiology of anabolism, feeding behavior, and nutritional homeostasis for GH secretion and gastrointestinal motility through gut–brain interactions. Other peptide hormones (i.e., motilin, TRH, PACAP, GnRH, leptin, FMRF amide, galanin, NPY, NPW) from gut, brain and other tissues also play a role in modulating GH secretion in livestock and lower vertebrate species. Physiological processes, such as neurotransmission, and secretion of hormones or enzymes, require fusion of secretory vesicles at the cell plasma membrane and expulsion of vesicular contents. This process for GH release from porcine somatotrophs was revealed by atomic force microscopy (AFM), transmission electron microscopy (TEM) and immunohistochemical distribution of the cells in pituitary during stages of development.

Circulating estradiol and hypothalamic corticotrophin releasing hormone enhances along with time after ovariectomy in rats: Effects of electroacupuncture

Repeated electroacupuncture (EA) stimulation is known to enhance the circulating level of estrogen in the ovariectomized rats by stimulating the activity of the hypothalamus-pituitary-adrenal axis. To explore the mechanism of EA in promoting the homeostasis in ovariectomized rats, the hypothalamic CRH content and its mRNA expression as well as the circulating estradiol in the rats along with time after ovariectomy were detected. Female rats were divided into fourteen groups: 1 month after ovariectomy (OVX1M), OVX2M, OVX3M, OVX4M, OVX5M, OVX6M; intact 1 month (INT1M), INT2M, INT3M, INT4M, INT5M, INT6M; OVX1M with EA (OVX1M+EA) and intact 1 month with EA (INT1M+EA). The blood concentrations of estradiol (E2) and total contents of hypothalamic CRH were measured by radioimmunoassay. The expressions of CRHmRNA were semiquantified by RT-PCR. The results showed that the blood E(2) concentrations increased gradually along with time after ovariectomy in the rats. The level was higher in OVX1M+EA than those in OVX1M, 2M, 3M (p < 0.05), which was near to the level in OVX6M. The total content (ng/g tissue) of hypothalamic CRH and the CRHmRNA expressions in the OVX6M rats were higher than other groups (p < 0.05). The CRH content or mRNA expression in the OVX1M+EA increased significantly compared with that in OVX1M (p < 0.05), and it was close to the level in the OVX4M and OVX5M. The results suggested that the circulating E2 and hypothalamic CRH enhances along with time after ovariectomy in rats, and the EA stimulation might speedup the process.

Neurochemical characterization of hypothalamic neurons involved in attack behavior: Glutamatergic dominance and co-expression of thyrotropin-releasing hormone in a subset of glutamatergic neurons

The electrical stimulation of a specific hypothalamic area rapidly evokes attacks in rats. Noteworthy, attack-related hypothalamic structures were identified in all species studied so far. The area has been extensively mapped in rats, and its anatomical connections have been studied in detail. However, technical difficulties precluded earlier the precise identification of the neural elements mediating the aggressive effects of stimulation. It now appears that a dense and distinct group of glutamatergic cells expressing vesicular glutamate transporter 2 mRNA extends over the entire hypothalamic attack area. Rostral parts overwhelmingly contained glutamatergic neurons. In more caudal parts, glutamatergic and fewer GABAergic neurons were found. The remarkable similarity in the distribution of hypothalamic attack area and glutamatergic cell groups suggests that these cells mediate the aggressive effects of stimulation. Surprisingly, thyrotropin releasing hormone mRNA was co-localized in a subset of glutamatergic neurons. Such neurons were present at all rostro-caudal levels of the hypothalamic attack area, except for that part of the hypothalamic attack area extending into the ventro-lateral part of the ventromedial hypothalamic nucleus. Earlier data on the projections of hypothalamic thyrotropin releasing hormone neurons suggest that this subpopulation plays a specific role in attack behavior. Thus, we identified three neuronal phenotypes in the hypothalamic structure that is involved in the induction of attacks: glutamatergic neurons co-expressing thyrotropin releasing hormone, glutamatergic neurons without thyrotropin releasing hormone, and GABAergic neurons dispersed among the glutamatergic cells. Assessing the specific roles and connections of these neuron subpopulations would contribute to our understanding of the mechanisms underlying attack behavior and aggression.

Multifunctional cells in human pituitary adenomas: implications for paradoxical secretion and tumorigenesis.

Pituitary adenomas are very common in humans. They are of monoclonal origin, very heterogeneous, and produce frequently paradoxical secretion. The normal anterior pituitary (AP) contains some unorthodox multifunctional cells able to store more than one AP hormone (polyhormonal) and/or to express multiple hypothalamic-releasing hormone receptors (multiresponsive). Multifunctional AP cells seem to be involved in plasticity processes such as transdifferentiation or paradoxical secretion. Here, we have characterized the single-cell phenotypes of 15 human pituitary tumors, including prolactinomas, nonfunctioning adenomas, and adenomas from multiple endocrine neoplasia type I (MEN-I) and pituitary Cushing's disease patients. Individual tumor cells were typed according to expression of AP hormones and hypothalamic-releasing hormone receptors by combination of calcium imaging and multiple sequential immunocytochemistry in the same cells. We found a large heterogeneity among the different tumors. In eight of the 15 tumors studied, more than 80% of the cells presented a multifunctional phenotype. This may explain the occurrence of paradoxical secretion. In addition, our results suggest that human pituitary adenomas might derive from multifunctional cells. This is consistent with the existence of a link between pituitary plasticity and tumorigenesis.

Anterior pituitary thyrotropes are multifunctional cells.

Anterior pituitary (AP) contains some unorthodox multifunctional cells that store and secrete two different AP hormones (polyhormonal cells) and/or respond to several hypothalamic-releasing hormones (HRHs; multiresponsive cells). Multifunctional cells may be involved in paradoxical secretion (secretion of a given AP hormone evoked by a noncorresponding HRH) and transdifferentiation (phenotypic switch between different mature cell types without cell division). Here we combine calcium imaging (to assess responses to the four HRHs) and multiple sequential immunoassay of the six AP hormones to perform a single-cell phenotypic study of thyrotropes in normal male and female mice. Surprisingly, most of the thyrotropes were polyhormonal, containing, in addition to thyrotropin (TSH), luteinizing hormone (40-42%) and prolactin (19-21%). Thyrotropes costoring growth hormone and/or ACTH were found only in females (24% of each type). These results suggest that costorage of the different hormones does not happen at random and that gender favors certain hormone combinations. Our results indicate that thyrotropes are a mosaic of cell phenotypes rather than a single cell type. The striking promiscuity of TSH storage should originate considerable mix-up of AP hormone secretions on stimulation of thyrotropes. However, response to thyrotropin-releasing hormone was much weaker in the polyhormonal thyrotropes than in the monohormonal ones. This would limit the appearance of paradoxical secretion under physiological conditions and suggests that timing of hormone and HRH receptor expression during the transdifferentiation process is finely and differentially regulated.

Receptors for hypothalamic releasing hormones TRH and GnRH: oligomerization and interactions with intracellular proteins.

Studies of TRH and GnRH receptors have revealed much information about the roles of G-proteins and beta-arrestins, as well as receptor residues important for signaling, desensitization and internalization. However, the proteins involved are only just beginning to be identified and characterized. Additional complexity now exists with the observation that these receptors form oligomers in live cells. Indeed, hetero-oligomerization of TRH receptor subtypes 1 and 2 potentially alters interactions with intracellular regulatory proteins. Knowledge of proteins that interact with TRH or GnRH receptors will increase our understanding of receptor function and provide potential drug targets for a range of receptor-associated conditions.

Dynamic contrast-enhanced MR imaging of the stimulated pituitary gland

Apoplexy due to infarction and/or hemorrhage is a frequent complication of pituitary adenoma, occurring either spontaneously or precipitated by several factors, among them pituitary function test with hypothalamic releasing hormones. The mechanism by which releasing hormones cause pituitary apoplexy is unclear. It has been proposed that increase in pituitary size and/or alterations in blood flow could be responsible. The aim of this study was to explore the effects of intravenous administration of hypothalamic releasing hormones on pituitary size and hemodynamics in healthy subjects. Gadolinium-DTPA-enhanced dynamic magnetic resonance imaging (MRI) was performed in eight healthy volunteers under basal conditions and 20 min after injection of releasing hormones. Mean upslopes of Gadolinium-DTPA enhancement curves showed good correlation between basal and stimulated conditions ( R = 0.89) and were significantly steeper after stimulation ( P = 0.017). In contrast, pituitary height, width and length did not differ significantly between basal and stimulated conditions. In conclusion, the pituitary does not swell in healthy subjects in response to stimulation with hypothalamic releasing hormones, whereas transfer of contrast agent to tissue (blood flow and/or vessel permeability) is enhanced.

Diagnostik des Wachstumshormonmangels und des Wachstumshormonexzesses (Akromegalie) / Diagnostics of growth hormone deficiency and excess (acromegaly)

Abstract Growth hormone (GH) is synthesized in the anterior lobe of the pituitary gland and has a plasma half-life of 19 minutes. GH-secretion is regulated by hypothalamic releasing hormone (GHRH) stimulation and somatostatin inhibition. The major effect of GH is the stimulation of insulin-like growth factor-1 (IGF-1) synthesis and secretion by the liver. However, GH also has other biological effects through other mediators. Serum-IGF-1 is bound to IGF binding protein-3 (IGFBP-3) and to acid-labile subunit (ALS). The GH/IGF-1-axis is evaluated when either GH-deficiency (GHD) or GH-excess (acromegaly) are suspected. GHD is confirmed by a diminished GHresponse to insulin-induced hypoglycemia (normal range GH &gt;5 μg/l) or to administration of GHRH+arginine (normal range GH &gt;9 μg/l). Acromegaly has distinct clinical signs and is confirmed when GH can not be suppressed below 1 μg/l by an oral glucose tolerance test in combination with elevated IGF-1 levels adjusted for age.

Active immunization against adrenocorticotrophic hormone (ACTH) in grazing lambs suppresses the immune response to a GnRH antigen but does not alter growth

This experiment examined the effects of immunizing against ACTH (adrenocorticotrophic hormone) and GnRH (hypothalamic peptide gonadotrophic releasing hormone) on growth and carcase composition of entire ram lambs from 6 to 35 weeks of age. There were four experimental groups (each of 17 animals) in a 2×2 factorial design: (1) a non-immunized control group, (2) immunized against ACTH, (3) immunized against GnRH and (4) immunized against both antigens. After booster injections, experimental lambs exhibited antibody titres to the two antigens presented, resulting in significantly lower cortisol and testosterone levels (P&lt;0·001) in peripheral blood. The fall in blood cortisol levels in response to ACTH immunization was later matched by a similar decline in control animals to a point where they were not different.In GnRH-immunized animals, antibodies disrupted the pituitary-gonadal axis, thereby impacting severely on testes development in growing lambs to the degree that by day 119 their testes were smaller than at the commencement of the experiment. Towards the end of the growth phase of the animals, GnRH-immunized lambs had significantly lower body weights at day 189 (P&lt;0·05) and day 204 (P&lt;0·01) while ACTH-immune lambs were not different in weight from control animals. Also, GnRH-immunized lambs were found to have greater GR measurement (P&lt;0·05). Although ACTH immunization was capable of lowering cortisol for a brief period during the current experiment, the results were variable and detrimental to the efficacy of the immuno-castration vaccine (GnRH).

Growth hormone secretion: molecular and cellular mechanisms and in vivo approaches.

Growth hormone (GH) release is under the direct control of hypothalamic releasing hormones, some being also produced peripherally. The role of these hypothalamic factors has been understood by in vitro studies together with such in vivo approaches as stalk sectioning. Secretion of GH is stimulated by GH-releasing hormone (GHRH) and ghrelin (acting via the GH secretagogue [GHS] receptor [GHSR]), and inhibited by somatostatin (SRIF). Other peptides/proteins influence GH secretion, at least in some species. The cellular mechanism by which the releasing hormones affect GH secretion from the somatotrope requires specific signal transduction systems (cAMP and/or calcium influx and/or mobilization of intracellular calcium) and/ or tyrosine kinase(s) and/or nitric oxide (NO)/cGMP. At the subcellular level, GH release (at least in response to GHS) is accomplished by the following. The GH-containing secretory granules are moved close to the cell surface. There is then transient fusion of the secretory granules with the fusion pores in the multiple secretory pits in the somatotrope cell surface.

Neuropeptidergic characterization of the leptin receptor mutated obese Koletsky rat

Leptin regulates energy homeostasis and reproduction as evidenced by dysfunctions characterized in several genetic models of leptin pathway deficiency, such as the ob/ob and db/db mice and fa/fa Zucker rat. An additional model, the obese ( f/f) Koletsky rat with a nonsense leptin receptor mutation has not been fully characterized. These rats are obese, hyperphagic, diabetic, and infertile; however, little else is known about the effects of the mutation. We have characterized alterations in hypothalamic appetite regulating neuropeptides as well as energy expenditure, metabolic hormones, and the reproductive axis of obese f/f rats. As expected, obese rats of both sexes were hyperinsulinemic, hyperglycemic, and hyperleptinemic. They exhibited reduced uncoupling protein-1 mRNA expression in brown fat, indicating reduced energy expenditure. In addition, hypothalamic expression of orexigenic neuropeptide Y and agouti-related peptide mRNA levels was upregulated while the anorexigenic cocaine and amphetamine regulated transcript and proopiomelanocortin mRNA levels were reduced. We also observed reproductive axis perturbations including reduced hypothalamic luteinizing hormone releasing hormone, serum estradiol and testosterone, and increased serum progesterone levels. In conclusion, obese Koletsky rats are phenotypically similar to other leptin pathway deficiency models with reduced energy expenditure and hypothalamic neuropeptidergic alterations that could account for their obesity and infertility.

Dynamic contrast – enhanced MR imaging of the stimulated pituitary gland

Apoplexy due to infarction and/or hemorrhage is a frequent complication of pituitary adenoma, occurring either spontaneously or precipitated by several factors, among them combined pituitary stimulation test with the four hypothalamic releasing hormones CRH, TRH, GHRH and GnRH. The mechanism by which releasing hormones cause pituitary apoplexy is unclear. It has been proposed that increase in pituitary size and/or alterations in blood flow could be responsible. The aim of this study was to explore the effects of intravenous administration of hypothalamic releasing hormones on pituitary size and hemodynamics in healthy subjects. Gadolinium-enhanced dynamic magnetic resonance imaging (MRI) was performed under basal conditions and 20 minutes after injection of the 4 hypothalamic releasing hormones. 2 out of 10 apparently healthy subjects had pituitary abnormalities in the basal MRI scan and were excluded from further analysis. In the remaining 8 subjects pituitary height, width and length were determined on corresponding MRI slices from basal and stimulated conditions. None of the measured parameters of pituitary size nor their product (as an additional surrogate of pituitary volume) differed significantly between basal and stimulated conditions. In contrast, mean upslopes of gadolinium enhancement curves were significantly steeper after stimulation (p=0.017). In conclusion, the pituitary does not swell in healthy subjects in response to stimulation with hypothalamic releasing hormones, whereas transfer of contrast agent to tissue (blood flow and/or vessel permeability) is enhanced.

Pituitary tumour clonality revisited.

Allelotype analysis and X chromosome inactivation analysis in women enables the assessment of tissue clonality, and has demonstrated that the majority of sporadic human pituitary adenomas are monoclonal. This implies that these tumours arise from de novo somatic genetic change(s) in a single pituitary cell. However, clonality within any given tumour may be multiple or single, multiple tumours arising on the background of hyperplasia may be of identical or different clonality, multiple 'sporadic' tumours in a tissue may be of differing clonal origin, and finally morphology cannot predict genetic makeup. These general principles may also apply to the pituitary so it is simplistic to assume that monoclonality is inevitable and that pituitary tumours cannot be multiclonal in origin. Indeed, these observations would be entirely compatible with the initiating stimulus resulting in hyperplasia of specific cell subtypes in the pituitary giving rise to a number of different clones each with variable potential to develop into a discrete tumour depending on their rate of cell division/rate of apotosis. Stimuli might include pituitary-specific oncogenes, intrapituitary growth factors, or extrapituitary trophic factors (e.g. hypothalamic releasing hormones).

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro

A bullfrog ( Rana catesbeiana) thyroid-stimulating hormone (TSH) β-subunit (TSHβ) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHβ cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHβ corresponded to those positively stained with an antiserum against human (h) TSHβ. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75 ± 0.07 ng TSH/100 μl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose–response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and α-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHβ coupled with the α-subunit by an immunoprecipitation experiment using the TSHβ antiserum and an α-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro. The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.

Absence of pituitary adenylate cyclase-activating polypeptide-stimulated transcription of the human glycoprotein alpha-subunit gene in LbetaT2 gonadotrophs reveals disrupted cAMP-mediated gene transcription.

Hormone regulation of anterior pituitary expression of the common glycoprotein hormone alpha-subunit (alphaGSU) is mediated by multiple response elements residing in the first -435 bp of the human promoter. In rat pituitary cells and mouse alphaT3-1 precursor gonadotrophs, the human alphaGSU promoter is strongly responsive to activators of the adenylyl cyclase/cAMP pathway, such as the hypothalamic releasing hormone, pituitary adenylate cyclase-activating polypeptide (PACAP) and forskolin (an adenylyl cyclase activator). However, the role of PACAP and cAMP in regulating alphaGSU transcription in the more differentiated LbetaT2 gonadotroph is unclear. Here, we investigate the regulation of the human alphaGSU promoter by PACAP and forskolin in LbetaT2 and alphaT3-1 gonadotrophs. PACAP failed to stimulate alphaGSU promoter activity or cAMP production in LbetaT2 cells, in marked contrast to alphaT3-1 cells. LbetaT2 gonadotrophs expressed extremely low levels of any PACAP type 1 receptors (PAC(1)-R) isoform by RT-PCR and lacked PAC(1)-R by radioligand binding. Forskolin stimulated the alphaGSU promoter in LbetaT2 cells, but by less than 30% of the response seen in alphaT3-1 gonadotrophs. This blunted cAMP transcriptional effect was not due to different levels of cAMP generation, or altered expression of the cAMP target proteins CREB, Akt, CBP or ICER. However, only LbetaT2 cells showed detectable expression of the protein kinase A type IIalpha regulatory subunit. Binding of activating transcription factor-2 and phosphorylated CREB to the consensus CRE was observed in both LbetaT2 and alphaT3-1 gonadotrophs, yet forskolin failed to stimulate either CRE- or CREB-mediated transcription in LbetaT2 cells. Collectively, these data demonstrate the lack of functional PACAP receptors in LbetaT2 gonadotrophs, and a pronounced attenuation in the responsiveness of this differentiated gonadotroph cell line to cAMP stimulus.

Engineering physiologically regulated insulin secretion in non-beta cells by expressing glucagon-like peptide 1 receptor.

Glucagon-like peptide 1 (GLP-1) is released from neuroendocrine cells in the intestine in the postprandial state and augments glucose-stimulated insulin secretion from pancreatic beta cells. To develop non-beta cells that exhibit physiologically regulated insulin secretion, we coexpressed the GLP-1 receptor and human insulin in primary rat pituitary cells using adenovirus-mediated gene transfer. The transduced cells were analyzed in a perifusion system and after transplantation into mice. Normal pituitary cells do not express the GLP-1 receptor as shown by the absence of GLP-1 receptor mRNA and the inability of GLP-1 to stimulate pituitary hormone secretion. Following transduction with an adenovirus carrying the GLP-1 receptor cDNA, the pituitary cells expressed functional GLP-1 receptors as reflected by the ability of GLP-1 to stimulate secretion of pituitary hormones. When both the GLP-1 receptor and human insulin were introduced, GLP-1 stimulated cosecretion of human insulin and endogenous pituitary hormones. GLP-1 was similar in potency to the hypothalamic-releasing hormones and stimulated hormone secretion in a dose-dependent fashion. In contrast to pancreatic beta cells, the hormone-releasing effect of GLP-1 on transduced pituitary cells was not dependent on the concentration of extracellular glucose. After transplantation of pituitary cells coexpressing human insulin and GLP-1 receptor into mice, enteral glucose stimulated insulin secretion. These results demonstrate a new approach to engineer physiologically regulated insulin secretion by non-beta cells.

Multifunctional cells of mouse anterior pituitary reveal a striking sexual dimorphism.

The existence of cells storing and secreting two different anterior pituitary (AP) hormones (polyhormonal cells) or responding to several hypothalamic releasing hormones (HRHs) (multiresponsive cells) has been reported previously. These multifunctional cells could be involved in paradoxical secretion (AP hormone secretion evoked by a non-corresponding HRH) and transdifferentiation (phenotypic switch between mature cell types without cell division). Despite their putative physiological relevance, a comprehensive characterization of multifunctional AP cells is lacking. Here we combine calcium imaging (to assess responses to the four HRHs) and multiple sequential immunoassay of the six AP hormones in the same individual cells to perform a complete phenotypic characterization of mouse AP cells. Polyhormonal and multiresponsive cells were identified within all five AP cell types. They were scarce in the more abundant cell types, somatotropes and lactotropes, but quite frequent in corticotropes and gonadotropes. Cells with mixed phenotypes were the rule rather than the exception in thyrotropes, where 56-83 % of the cells stored two to five different hormones. Multifunctional AP cells were much more abundant in females than in males, indicating that the hormonal changes associated with the sexual cycle may promote transdifferentiation. As the phenotypic analysis was performed here after stimulation with HRHs, the fraction of polyhormonal cells might have been underestimated. With this limitation, the polyhormonal cells detected here responded to the HRHs less than the monohormonal ones, suggesting that they might contribute less than expected a priori to paradoxical secretion. Overall, our results reveal a striking sexual dimorphism, the female pituitary being much more plastic than the male pituitary.