Novel pituitary actions of TAC3 gene products in fish model: Receptor specificity and signal transduction for prolactin and somatolactin α regulation by neurokinin B (NKB) and NKB-related peptide in carp pituitary cells

TAC3 is a member of tachykinins, and its gene product neurokinin B (NKB) has recently emerged as a key regulator for LH through modulation of kisspeptin/GnRH system within the hypothalamus. In fish models, TAC3 not only encodes NKB but also a novel tachykinin-like peptide called NKB-related peptide (NKBRP), and the pituitary actions of these TAC3 gene products are still unknown. Using grass carp as a model, the direct effects and postreceptor signaling for the 2 TAC3 products were examined at the pituitary level. Grass carp TAC3 was cloned and confirmed to encode NKB and NKBRP similar to that of other fish species. In carp pituitary cells, NKB and NKBRP treatment did not affect LH release and gene expression but up-regulated prolactin (PRL) and somatolactin (SL)α secretion, protein production, and transcript expression. The stimulation by these 2 TAC3 gene products on PRL and SLα release and mRNA levels were mediated by pituitary NK2 and NK3 receptors, respectively. Apparently, NKB- and NKBRP-induced SLα secretion and transcript expression were caused by adenylate cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C and Ca(2+)/calmodulin/Ca(2+)/calmodulin-dependent protein kinase II activation. The signal transduction for the corresponding responses on PRL release and mRNA expression were also similar, except that the protein kinase C component was not involved. These findings suggest that the 2 TAC3 gene products do not play a role in LH regulation at the pituitary level in carp species but may serve as novel stimulators for PRL and SLα synthesis and secretion via overlapping postreceptor signaling mechanisms coupled to NK2 and NK3 receptors, respectively.postprin

Dual role of insulin in spexin regulation: Functional link between food intake and spexin expression in fish model

Spexin (SPX), a neuropeptide discovered by the bioinformatics approach, has been recently identified as a satiety factor in a fish model. However, the functional link between feeding and SPX expression as well as the signal transduction for SPX regulation are totally unknown. In this study, we used goldfish as a model to examine the functional role of insulin as a postprandial signal for SPX regulation in bony fish. In goldfish, feeding could elevate plasma levels of glucose, insulin, and SPX with concurrent rises in insulin and SPX messenger RNA (mRNA) expression in the liver. Similar elevation in SPX mRNA level was also observed in the liver and brain areas involved in appetite control in goldfish after intraperitoneal injection of glucose and insulin, respectively. In parallel experiments with goldfish hepatocytes and brain cell culture, insulin signal induced by glucose was shown to exert a dual role in SPX regulation, namely (1) acting as an autocrine/paracrine signal to trigger SPX mRNA expression in the liver and (2) serving as an endocrine signal to induce SPX gene expression in the brain. Apparently, the peripheral (in the liver) and central actions of insulin (in the brain) on SPX gene expression were mediated by insulin receptor (to a lesser extent by insulin-like growth factor I receptor) coupled to mitogen-activated protein kinase kinase 3/6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin but not mitogen-activated protein kinase kinase 1/2/extracellular signal-regulated kinase 1/2 cascades. Our findings indicate that an insulin component inducible by glucose is present in the liver of the fish model and may serve as the postprandial signal linking food intake with SPX expression both in the central as well as at the hepatic level.postprin

Insulin-like growth factor as a novel stimulator for somatolactin secretion and synthesis in carp pituitary cells via activation of MAPK cascades

Somatolactin (SL), a member of the growth hormone/prolactin family, is a pituitary hormone unique to fish models. Although SL is known to have diverse functions in fish, the mechanisms regulating its secretion and synthesis have not been fully characterized. Using grass carp pituitary cells as a model, here we examined the role of insulin-like growth factor (IGF) in SL regulation at the pituitary level. As a first step, the antisera for the two SL isoforms expressed in the carp pituitary, SLα and SLβ, were produced, and their specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the carp pituitary. Western blot using these antisera revealed that grass carp SLα and SLβ could be N-linked glycosylated and their basal secretion and cell content in carp pituitary cells could be elevated by IGF-I and -II treatment. These stimulatory effects occurred with parallel rises in SLα and SLβ mRNA levels, and these SL gene expression responses were not mimicked by insulin but blocked by IGF-I receptor inactivation. In carp pituitary cells, IGF-I and -II could induce rapid phosphorylation of IGF-I receptor, MEK1/2, ERK1/2, MKK3/6, and p38 MAPK; and SLα and SLβ secretion, protein production, and mRNA expression caused by IGF-I and -II stimulation were negated by inactivating MEK1/2 and p38 MAPK. Parallel inhibition of PI3K and Akt, however, were not effective in these regards. These results, taken together, provide evidence that IGF can upregulate SL secretion and synthesis at the pituitary level via stimulation of MAPK- but not PI3K/Akt-dependent pathways. © 2011 the American Physiological Society.link_to_subscribed_fulltex

Feedback regulation of growth hormone synthesis and secretion in fish and the emerging concept of intrapituitary feedback loop

Growth hormone (GH) is known to play a key role in the regulation of body growth and metabolism. Similar to mammals, GH secretion in fish is under the control of hypothalamic factors. Besides, signals generated within the pituitary and/or from peripheral tissues/organs can also exert a feedback control on GH release by effects acting on both the hypothalamus and/or anterior pituitary. Among these feedback signals, the functional role of IGF is well conserved from fish to mammals. In contrast, the effects of steroids and thyroid hormones are more variable and appear to be species-specific. Recently, a novel intrapituitary feedback loop regulating GH release and GH gene expression has been identified in fish. This feedback loop has three functional components: (i) LH induction of GH release from somatotrophs, (ii) amplification of GH secretion by GH autoregulation in somatotrophs, and (iii) GH feedback inhibition of LH release from neighboring gonadotrophs. In this article, the mechanisms for feedback control of GH synthesis and secretion are reviewed and functional implications of this local feedback loop are discussed. This intrapituitary feedback loop may represent a new facet of pituitary research with potential applications in aquaculture and clinical studies. © 2005 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex

Grass carp somatolactin: I. Evidence for PACAP induction of somatolactin-α and -β gene expression via activation of pituitary PAC-I receptors

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. At present, SL can be identified only in fish but not in tetrapods and its regulation at the pituitary level has not been fully characterized. Using grass carp as a model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL secretion and synthesis at the pituitary cell level. As a first step, the structural identity of grass carp SL, SLα and SLβ, was established by 5′/3′-rapid amplification of cDNA ends. These two SL isoforms are single-copy genes and are expressed in two separate populations of pituitary cells located in the pars intermedia. In the carp pituitary, PACAP nerve fibers were detected in the nerve tracts of the neurohypophysis and extended into the vicinity of pituitary cells forming the pars intermedia. In primary cultures of grass carp pituitary cells, PACAP was effective in stimulating SL release, cellular SL content, and total SL production. The increase in SL production also occurred with parallel rises in SLα and SLβ mRNA levels. With the use of a combination of molecular and pharmacological approaches, PACAP-induced SL release and SL gene expression were shown to be mediated by pituitary PAC-I receptors. These findings, as a whole, suggest that PACAP may serve as a hypophysiotropic factor in fish stimulating SL secretion and synthesis at the pituitary level. Apparently, PACAP-induced SL production is mediated by upregulation of SLα and SLβ gene expression through activation of PAC-I receptors. Copyright © 2008 the American Physiological Society.link_to_subscribed_fulltex

Novel aspects of growth hormone (GH) autoregulation: GH-induced GH gene expression in grass carp pituitary cells through autocrine/paracrine mechanisms

GH feedback on its own secretion at the pituitary level has been previously reported, but the mechanisms involved have not been elucidated. Here we examined the autocrine/paracrine effects of GH on GH synthesis using grass carp pituitary cells as a cell model. GH receptors were identified in carp somatotrophs, and their activation by exogenous GH increased steady-state GH mRNA levels and GH production. Removal of endogenous GH by immunoneutralization using GH antiserum inhibited basal as well as stimulated GH mRNA expression induced by GH-releasing factors in fish, including GnRH, apamorphine, and pituitary adenylate cyclase-activating polypeptide-38. Cytosolic mature GH mRNA levels were elevated by GH treatment and reduced by GH antiserum, whereas nuclear GH primary transcripts were almost undetectable after GH immunoneutralization. Inhibition of Janus kinase-2 (JAK 2), phosphoinositide 3-kinase, and MAPK also abolished GH-induced steady-state GH mRNA expression. GH immunoneutralization in pituitary cells pretreated with actinomycin D induced a marked decrease in the half-life of GH mRNA, indicating that the clearance of GH transcripts could be enhanced by removing endogenous GH. These results provide evidence that GH can serve as a novel intrapituitary autocrine/paracrine factor maintaining GH gene expression in somatotrophs, and this action is mediated by JAK 2/MAPK and JAK 2/ phosphoinositide 3-kinase cascades coupled to GH receptors.link_to_subscribed_fulltex

Goldfish kisspeptin: Molecular cloning, tissue distribution of transcript expression, and stimulatory effects on prolactin, growth hormone and luteinizing hormone secretion and gene expression via direct actions at the pituitary level

Kisspeptin, the product of Kiss1 gene, is a novel regulator of the gonadotropic axis. In mammals, its stimulatory effect on gonadotropin secretion is well documented and mediated mainly by hypothalamic release of gonadotropin-releasing hormone. Although the pituitary actions of kisspeptin have been reported, the effects of kisspeptin on gonadotropin release via direct action on pituitary cells are still controversial. Using goldfish as a model, here we examined the direct actions of kisspeptin on pituitary functions in modern-day bony fish. As a first step, the structural identity of goldfish Kiss1 was established by 5′/3′RACE and Kiss1 transcript was shown to be widely expressed in various tissues in goldfish. At the pituitary level, Kiss1 receptor (Kiss1r) expression was detected in immuno-identified gonadotrophs, lactotrophs, and somatotrophs. Kiss1 transcript was also located in goldfish somatotrophs but not in lactotrophs or gonadotrophs. In parallel studies, goldfish kisspeptin-10 was synthesized and used to test the pituitary actions of kisspeptin in vitro. In goldfish pituitary cell cultures, 30-min incubation with kisspeptin-10 increased basal release of luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH). Transcript expression of LH, PRL, and GH were also elevated by prolonging kisspeptin-10 treatment to 24 h. These results taken together suggest that kisspeptin via Kiss1r activation can act directly at the pituitary level to trigger LH, PRL, and GH secretion and gene expression in goldfish. Our finding of Kiss1 expression in somatotrophs also rises the possibility that kisspeptin may be produced locally in the fish pituitary and serve as an autocrine/paracrine regulator. © 2009 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex

Modulation of calmodulin gene expression as a novel mechanism for growth hormone feedback control by insulin-like growth factor in grass carp pituitary cells

Calmodulin (CaM), the Ca2+ sensor in living cells, is essential for biological functions mediated by Ca2+-dependent mechanisms. However, modulation of CaM gene expression at the pituitary level as a means to regulate pituitary hormone synthesis has not been characterized. Here we examined the functional role of CaM in the feedback control of growth hormone (GH) by insulin-like growth factor (IGF) using grass carp pituitary cells as a cell model. To establish the structural identity of CaM expressed in the grass carp, a CaM cDNA, CaM-L, was isolated from the carp pituitary using 3′/5′ RACE. The ORF of this cDNA encodes a 149 a.a. protein sharing the same primary structure with CaMs reported in mammals, birds, and amphibians. This CaM cDNA is phylogenetically related to the CaM I gene family and its transcripts are ubiquitously expressed in the grass carp. In carp pituitary cells, IGF-I and -II induced CaM mRNA expression with a concurrent drop in GH transcript levels. These stimulatory effects on CaM mRNA levels were not mimicked by insulin and appeared to be a direct consequence of IGF activation of CaM gene transcription without altering CaM transcript stability. CaM antagonism and inactivation of calcineurin blocked the inhibitory effects of IGF-I and -II on GH gene expression and CaM over-expression also suppressed the 5′ promoter activity of grass carp GH gene. These results, as a whole, provide evidence for the first time that IGF feedback on GH gene expression is mediated by activation of CaM gene expression at the pituitary level. Copyright © 2005 by The Endocrine Society.link_to_subscribed_fulltex

Paracrine regulation of growth hormone gene expression by gonadotrophin release in grass carp pituitary cells: Functional implications, molecular mechanisms and signal transduction

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased 'steady-state' GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44 MAPK and P38 MAPK. These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway. © 2005 Society for Endocrinology.link_to_subscribed_fulltex

A territory-wide study on the seroprevalence of hepatitis B virus in the general population after universal hepatitis B immunization era in Hong Kong, China

Poster Presentations: Viral hepatitis: Hepatitis A, B, D, E – clinical (except therapy): no. THU-162BACKGROUND AND AIMS: Chronic hepatitis B (CHB) remains a major health burden in the Asia-Pacific region. No population-based study has been carried out to assess the territorial prevalence of CHB after the introduction of universal hepatitis B vaccination program in1988 in Hong Kong. We aimed to document the point prevalence of viral hepatitis B in Hong Kong …Link_to_subscribed_fulltex