Caleb L. Grey

Signal transduction in multifactorial neuroendocrine control of gonadotropin secretion and synthesis in teleosts-studies on the goldfish model.

In teleosts, gonadotropin (GTH) secretion and synthesis is controlled by multiple neuroendocrine factors from the hypothalamus, pituitary and peripheral sources. Pituitary gonadotropes must be able to differentiate and integrate information from these regulators at the cellular and intracellular level. In this article, the intracellular signal transduction mechanisms mediating the actions of some of these regulators, including GTH-releasing hormones, pituitary adenylate cyclase-activating polypeptide, dopamine, ghrelin, sex steroids, activin, and follistatin from experiments with goldfish are reviewed and discussed in relation with recent findings. Information from other teleost models is briefly compared. Goldfish gonadotropes possess multiple pharmacologically distinct intracellular Ca2+ stores that together with voltage-sensitive Ca2+ channels, Na+/H+ exchangers, protein kinase C, arachidonic acid, NO, protein kinase A, ERK/MAPK, and Smads allows for integrated control by different neuroendocrine factors.

Transcript Expression Data from Human Islets Links Regulatory Signals from Genome-Wide Association Studies for Type 2 Diabetes and Glycemic Traits to Their Downstream Effectors.

The intersection of genome-wide association analyses with physiological and functional data indicates that variants regulating islet gene transcription influence type 2 diabetes (T2D) predisposition and glucose homeostasis. However, the specific genes through which these regulatory variants act remain poorly characterized. We generated expression quantitative trait locus (eQTL) data in 118 human islet samples using RNA-sequencing and high-density genotyping. We identified fourteen loci at which cis-exon-eQTL signals overlapped active islet chromatin signatures and were coincident with established T2D and/or glycemic trait associations. ‎At some, these data provide an experimental link between GWAS signals and biological candidates, such as DGKB and ADCY5. At others, the cis-signals implicate genes with no prior connection to islet biology, including WARS and ZMIZ1. At the ZMIZ1 locus, we show that perturbation of ZMIZ1 expression in human islets and beta-cells influences exocytosis and insulin secretion, highlighting a novel role for ZMIZ1 in the maintenance of glucose homeostasis. Together, these findings provide a significant advance in the mechanistic insights of T2D and glycemic trait association loci.

Calcium and other signalling pathways in neuroendocrine regulation of somatotroph functions

Relative to mammals, the neuroendocrine control of pituitary growth hormone (GH) secretion and synthesis in teleost fish involves numerous stimulatory and inhibitory regulators, many of which are delivered to the somatotrophs via direct innervation. Among teleosts, how multifactorial regulation of somatotroph functions are mediated at the level of post-receptor signalling is best characterized in goldfish. Supplemented with recent findings, this review focuses on the known intracellular signal transduction mechanisms mediating the ligand- and function-specific actions in multifactorial control of GH release and synthesis, as well as basal GH secretion, in goldfish somatotrophs. These include membrane voltage-sensitive ion channels, Na(+)/H(+) antiport, Ca(2+) signalling, multiple pharmacologically distinct intracellular Ca(2+) stores, cAMP/PKA, PKC, nitric oxide, cGMP, MEK/ERK and PI3K. Signalling pathways mediating the major neuroendocrine regulators of mammalian somatotrophs, as well as those in other major teleost study model systems are also briefly highlighted. Interestingly, unlike mammals, spontaneous action potential firings are not observed in goldfish somatotrophs in culture. Furthermore, three goldfish brain somatostatin forms directly affect pituitary GH secretion via ligand-specific actions on membrane ion channels and intracellular Ca(2+) levels, as well as exert isoform-specific action on basal and stimulated GH mRNA expression, suggesting the importance of somatostatins other than somatostatin-14.

Nesfatin-1 Regulates the Hypothalamo-Pituitary-Ovarian Axis of Fish

ABSTRACT Nesfatin-1 is an anorexigen in goldfish. In the present study, we provide novel data indicating the presence and regulatory effects of nesfatin-1 on the hypothalamo-pituitary-ovarian (HPO) axis of goldfish. Nucleobindin-2 (NUCB2)/nesfatin-1-like immunoreactive (ir) cells are present in the hypothalamus and in the pituitary, suggesting a hypophysiotropic role for nesfatin-1. NUCB2/nesfatin-1-like ir cells colocalize gonadotropin-releasing hormone (GnRH) in the nucleus lateralis tuberis posterioris and the nucleus anterior tuberis of the goldfish hypothalamus. The presence of nesfatin-1 with GnRH in these two nuclei implicated in pituitary hormone release suggests a role for nesfatin-1 on gonadotropin secretion. A single i.p. injection of synthetic goldfish nesfatin-1 (50 ng/g body wt) resulted in an acute decrease (∼75%) in the expression of hypothalamic chicken GnRH-II and salmon GnRH mRNAs at 15 min postinjection in goldfish. Meanwhile, pituitary luteinizing hormone (LH) beta and follicle-stimulating hormone beta mRNAs were also inhibited (∼80%), but only at 60 min postinjection. Nesfatin-1 administration also resulted in a significant reduction (∼60%) in serum LH levels at 60 min postadministration. Nesfatin-1-like immunoreactivity was also found in the follicle cells, but not the oocytes, in zebrafish and goldfish ovaries. Incubation of zebrafish follicles with nesfatin-1 resulted in a significant reduction in basal germinal vesicle breakdown (∼50%) during the oocyte maturation. In addition, nesfatin-1 also attenuated the stimulatory effects of maturation-inducing hormone on germinal vesicle breakdown. Together, the current results indicate that nesfatin-1 is a metabolic hormone with an inhibitory tone on fish reproduction. Nesfatin-1 appears to elicit this suppressive effect through actions on all three tissues in the fish HPO axis.

The secretogranin II-derived peptide secretoneurin stimulates luteinizing hormone secretion from gonadotrophs.

Secretoneurin (SN) is a 33- to 34-amino acid neuropeptide derived from secretogranin-II, a member of the chromogranin family. We previously synthesized a putative goldfish (gf) SN and demonstrated its ability to stimulate LH release in vivo. However, it was not known whether goldfish actually produced the free SN peptide or whether SN directly stimulates LH release from isolated pituitary cells. Using a combination of reverse-phase HPLC and mass spectrometry analysis, we isolated for the first time a 34-amino acid free gfSN peptide from the whole brain. Moreover, Western blot analysis indicated the existence of this peptide in goldfish pituitary. Immunocytochemical localization studies revealed the presence of SN immunoreactivity in prolactin cells of rostral pars distalis of the anterior pituitary. Additionally, we found that magnocellular cells of the goldfish preoptic region are highly immunoreactive for SN. These neurons send heavily labeled projections that pass through the pituitary stalk and innervate the neurointermediate and anterior lobes. In static 12-h incubation of dispersed pituitary cells, application of SN antiserum reduced LH levels, whereas 1 and 10 nM gfSN, respectively, induced 2.5-fold (P < 0.001) and 1.9-fold (P < 0.01) increments of LH release into the medium, increases similar to those elicited by 100 nM concentrations of GnRH. Like GnRH, gfSN elevated intracellular Ca(2+) in identified gonadotrophs. Whereas we do not yet know the relative contribution of neural SN or pituitary SN to LH release, we propose that SN could act as a neuroendocrine and/or paracrine factor to regulate LH release from the anterior pituitary.

Ghrelin-induced growth hormone release from goldfish pituitary cells involves voltage-sensitive calcium channels.

Ghrelin (GRL) is a stimulator of growth hormone (GH) release in many organisms, including goldfish. As a first study to examine the signalling mechanisms mediating GRL action on GH release in goldfish, we tested the hypothesis that GLR induces GH release from goldfish pituitary cells by enhancing Ca(2+) entry through L-type voltage-sensitive Ca(2+) channels (LVSCCs) using perifusion GH release and fura-2/AM Ca(2+)-imaging experiments. Goldfish (g)GRL(19) at 1 nM elicited reversible and repeatable GH responses from dispersed goldfish mixed pituitary cultures. However, the lack of a dose-response relationship in sequential treatments with decreasing concentrations of gGRL(19) (ranging from 10 to 0.01 nM) implicated rapid desensitization of the GH response. Sequential applications of gGRL(19) (1 nM) and salmon GnRH (100 nM), a known Ca(2+)-dependent stimulator of GH release, increased intracellular free Ca(2+) levels ([Ca(2+)](i)) from the same identified somatotropes, suggesting co-expression of GRL and GnRH receptors on single cells. In contrast, 1 nM gGRL(19) failed to elicit GH release and elevation in [Ca(2+)](i) when the cells are incubated with nominally Ca(2+)-free media. When GH release and [Ca(2+)](i) increases were already stimulated by the LVSCC agonist Bay K8644 (10 microM), addition of 1 nM gGRL(19) did not further elevate these responses. Finally, the LVSCC inhibitors nifedipine (1 microM) and verapamil (1 microM) abolished 1nM gGRL(19)-induced GH release responses while nifedipine eliminated gGRL(19)-induced [Ca(2+)](i) increase. Taken together, the results of this study provide evidence that entry of extracellular Ca(2+) through LVSCCs is a key component of the GRL signalling pathway leading to GH release in the goldfish pituitary.

Ghrelin stimulation of gonadotropin (LH) release from goldfish pituitary cells: presence of the growth hormone secretagogue receptor (GHS-R1a) and involvement of voltage-sensitive Ca2+ channels.

Ghrelin (GRLN) stimulates maturational gonadotropin (LH) secretion in goldfish. This study identified GRLN receptors (GHS-Rs) in goldfish tissues and examined the involvement of voltage-sensitive Ca(2+) channels (VSCCs) in ghrelin action. A partial goldfish GHS-R1a sequence was obtained and expression observed in brain, pituitary, spleen, kidney, heart, gill, ovary, testis, and intestine. Synthetic goldfish GRLN (gGRLN(19)) stimulated LH secretion from dispersed goldfish pituitary cells in column perifusion and increased intracellular Ca(2+) ([Ca(2+)](i)) in identified goldfish gonadotropes. gGRLN(19) did not stimulate LH secretion either in the presence of Ca(2+)-free media, or the L-type VSCC inhibitors nifedipine and verapamil. Similarly, gGRLN(19)-elicited increases in [Ca(2+)](i) were attenuated by Ca(2+)-free media and nifedipine. Furthermore, when LH release and [Ca(2+)](i) were elevated by Bay K8644, gGRLN(19) had no further effect. These results indicate that GHS-R1a is present in goldfish pituitary and Ca(2+) entry through VSCC mediates direct gGRLN(19) action on LH release in goldfish pituitary cells.

Growth hormone-releasing hormone stimulates GH release while inhibiting ghrelin- and sGnRH-induced LH release from goldfish pituitary cells

Goldfish GH-releasing hormone (gGHRH) has been recently identified and shown to stimulate GH release in goldfish. In goldfish, neuroendocrine regulation of GH release is multifactorial and known stimulators include goldfish ghrelin (gGRLN19) and salmon gonadotropin-releasing hormone (sGnRH), factors that also enhance LH secretion. To further understand the complex regulation of pituitary hormone release in goldfish, we examined the interactions between gGHRH, gGRLN19, and sGnRH on GH and LH release from primary cultures of goldfish pituitary cells in perifusion. Treatment with 100nM gGHRH for 55min stimulated GH release. A 5-min pulse of either 1nM gGRLN19 or 100nM sGnRH induced GH release in naïve cells, and these were just as effective in cells receiving gGHRH. Interestingly, gGHRH abolished both gGRLN19- and sGnRH-induced LH release and reduced basal LH secretion levels. These results suggest that gGHRH does not interfere with sGnRH or gGRLN19 actions in the goldfish somatotropes and further reveal, for the first time, that GHRH may act as an inhibitor of stimulated and basal LH release by actions at the level of pituitary cells.

Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins (R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression (R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca(2+) concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

Ghrelin-induced growth hormone release from goldfish pituitary cells is nitric oxide dependent

Ghrelin (GRLN) is an important neuroendocrine regulator of growth hormone (GH) release in vertebrates. Previous studies show goldfish (g)GRLN(19)-induced GH from the goldfish pituitary involves voltage sensitive Ca(2+) channels, increases in intracellular Ca(2+) and the PKC signalling pathway. We set out to examine the role of the nitric oxide (NO) pathway in gGLRN(19)-induced GH release from primary cultures of goldfish pituitary cells using pharmacological regulators in cell column perifusion systems. The NO scavenger PTIO abolished gGRLN(19)-induced GH release and co-treatment with the NO donor SNP and GRLN did not produce additive GH release responses. Nitric oxide synthase (NOS) inhibitors 1400 W and 7-Ni abolished GRLN-induced GH release while treatment with another NOS inhibitor, AGH, had no significant effect. Taken together, these results demonstrate that the NOS/NO is an integral component of gGRLN(19)-induced signalling within the goldfish pituitary cells, and given the relative specificity of AGH for inducible NOS and endothelial NOS isoforms, suggests that neuronal NOS is the likely NOS isoform utilized in goldfish somatotropes by this physiological regulator.