Pituitary Cell Cultures Research Articles

Differential in vitro response to octreotide and pasireotide in normal and tumoral primary pituitary cell cultures

Differential in vitro response to octreotide and pasireotide in normal and tumoral primary pituitary cell cultures

Corticotropin-releasing hormone and the sympathoadrenal system are major mediators in the effects of peripherally administered exendin-4 on the hypothalamic-pituitary-adrenal axis of male rats.

Glucagon-like peptide-1 (GLP-1) and the GLP-1 receptor agonist, exendin-4 (Ex-4), potently stimulate hypothalamic-pituitary-adrenal (HPA) axis activity after either central or peripheral administration. Because several GLP-1 derivative drugs, including synthetic Ex-4, are currently in use to treat patients with type II diabetes mellitus, the characterization of Ex-4 effects on the HPA axis is highly relevant. Herein, the roles of CRH and AVP on these effects were investigated by administering the antagonists astressin and d(CH2)5Tyr(Me)AVP, respectively. The role of the sympathoadrenal system (SAS) was explored in bilateral adrenal enucleated and guanethidine-treated rats, whereas primary pituitary cell cultures were used to study direct effects on the corticotropes. Astressin completely abrogated (P < .05) the effects of Ex-4 central administration on ACTH secretion but only slightly reduced (by 35%) the ACTH response to Ex-4 peripheral administration. Moreover, astressin significantly (P < .05) decreased the corticosterone response to centrally but not peripherally administered Ex-4, suggesting different mechanisms depending on the route of administration. Pretreatment with d(CH2)5Tyr(Me)AVP failed to diminish either the ACTH or corticosterone response to Ex-4 and no direct effect of Ex-4 or GLP-1 was observed on pituitary cell cultures. In contrast, a significant (P < .05) reduction in the corticosterone response elicited by Ex-4 peripheral administration was observed in enucleated and guanethidine-treated rats, indicating a role of the SAS in the glucocorticoid stimulatory effects of Ex-4. Our data demonstrate that the effects of Ex-4 on the HPA axis are partially mediated by CRH and the sympathoadrenal system, and stress the relevance of Ex-4 as a corticosterone secretagogue.

Inflammatory triggers of angiogenic factor production in pituitary tumor cells

In pituitary adenomas, which are known to display decreased vascular density in comparison to normal pituitary tissue, the exact role of angiogenesis in tumor transformation and progression remains unclear. As our previous studies performed on pituitary adenoma cell cultures have shown an increased basal production of the pro-angiogenic and pro-inflammatory cytokine IL-8 compared to normal pituitaries, we have chosen to take a closer look at the possible role of inflammation-modulated angiogenesis in pituitary adenomas.

Regulation of LH/FSH expression by secretoglobin 3A2 in the mouse pituitary gland

Secretoglobin (SCGB) 3A2 was originally identified as a downstream target for the homeodomain transcription factor NKX2-1 in the lung. NKX2-1 plays a role in the genesis and expression of genes in the thyroid, lung and ventral forebrain; Nkx2-1-null mice have no thyroid and pituitary and severely hypoplastic lungs and hypothalamus. To demonstrate whether SCGB3A2 plays any role in pituitary hormone production, NKX2-1 and SCGB3A2 expression in the mouse pituitary gland was examined by immunohistochemical analysis and RT-PCR. NKX2-1 was localized in the posterior pituitary lobe, whereas SCGB3A2 was observed in both anterior and posterior lobes as shown by immunohistochemistry and RT-PCR. Expression of CCAAT-enhancer binding proteins (C/EBPs), which regulate mouse Scgb3a2 transcription, was also examined by RT-PCR. C/EBPβ, γ, δ and ζ were expressed in the adult mouse pituitary gland. SCGB3A2 was expressed in the anterior and posterior lobes from postnatal days 1 and 5, respectively and the areas where SCGB3A2 expression was found coincided with the area where FSH-secreting cells were found. Double-staining for SCGB3A2 and pituitary hormones revealed that SCGB3A2 was mainly localized in gonadotrophs in 49 % of FSH-secreting cells and 47 % of LH-secreting cells. In addition, SCGB3A2 dramatically inhibited LH and FSH mRNA expression in rat pituitary primary cell cultures. These results suggest that SCGB3A2 regulates FSH/LH production in the anterior pituitary lobe and that transcription factors other than NKX2-1 may regulate SCGB3A2 expression.

Obestatin Plays an Opposite Role in the Regulation of Pituitary Somatotrope and Corticotrope Function in Female Primates and Male/Female Mice

Obestatin is a 23-amino-acid amidated peptide that is encoded by the ghrelin gene. Previous studies have shown obestatin can modulate the hypothalamic neuronal circuitry that regulates pituitary function, perhaps by modulating the actions of ghrelin. However, the direct actions of obestatin on pituitary function remain controversial. Here, primary pituitary cell cultures from a nonhuman primate (baboon) and mice were used to test the effects of obestatin on pituitary hormone expression and secretion. In pituitary cultures from both species, obestatin had no effect on prolactin, LH, FSH, or TSH expression/release. Conversely, obestatin stimulated proopiomelanocortin expression and ACTH release and inhibited GH expression/release in vitro, actions that were also observed in vivo in mice treated with obestatin. In vitro, obestatin inhibited the stimulatory actions of ghrelin on GH but not ACTH release. The inhibitory effect of obestatin on somatotrope function was associated with an overall reduction in pituitary transcription factor-1 and GHRH receptor mRNA levels in vitro and in vivo as well as a reduction in hypothalamic GHRH and ghrelin expression in vivo. The stimulatory effect of obestatin on ACTH was associated with an increase in pituitary CRF receptors. Obestatin also reduced the expression of pituitary somatostatin receptors (sst1/sst2), which could serve to modify its impact on hormone secretion. The in vitro actions of obestatin on both GH and ACTH release required the adenylyl cyclase and MAPK routes. Taken together, our results provide evidence that obestatin can act directly at the pituitary to control somatotrope and corticotrope function, and these effects are conserved across species.

Trichostatin A specifically stimulates gonadotropin FSHβ gene expression in gonadotroph LβT2 cells

Trichostatin A (TSA) is a selective inhibitor of mammalian histone deacetylase. In the present study, TSA was found to selectively increase gene expression of the pituitary gonadotropin β-subunit of follicle-stimulating hormone (FSH). Stimulation of mouse pituitary gonadotroph cell lines, LβT2, with TSA for 24 h resulted in no change in mRNA expression of the α- and LHβ-subunit. On the other hand, FSHβ-subunit mRNA expression was significantly increased in a dose-dependent fashion. Similarly, specific induction of the FSHβ-subunit gene with TSA stimulation was observed in primary cultures of rat pituitary cells. Histone acetylation in whole cell lysates of LβT2 cells was significantly increased after TSA treatment, but not gonadotropin-releasing hormone (GnRH) treatment. The effect of TSA on FSHβ mRNA expression was prominent compared to that of GnRH; however, TSA-stimulated FSHβ mRNA expression was significantly reduced with combined TSA and GnRH treatment. TSA caused a slight increase in extracellular signal-regulated kinase (ERK) phosphorylation, while GnRH-increased ERK phosphorylation was potentiated in the presence of TSA. In addition, TSA, but not GnRH, significantly stimulated gene expression of retinaldehyde dehydrogenase 1 (RALDH1), a retinoic acid (RA) synthesizing enzyme involved in cell differentiation. These findings demonstrate that TSA specifically increases FSHβ subunit gene expression with a concomitant increase in whole cell histone acetylation. Moreover, although GnRH is a stimulator of FSHβ gene expression, it interfered with the stimulatory effect of TSA on FSHβ mRNA expression, without modification of TSA-increased whole cell histone acetylation. This suggests that the mechanisms of TSA and GnRH-induced gonadotropin subunit gene expression are entirely distinct.

Expression of gonadotropin-inhibitory hormone receptors in mouse pituitary gonadotroph LβT2 cells and hypothalamic gonadotropin-releasing hormone-producing GT1-7 cells

Gonadotropin-inhibitory hormone (GnIH) was first identified in quail as a novel neurohormone that acts directly on the anterior pituitary to inhibit gonadotropin release. GnIH inhibits not only gonadotropin release from the pituitary gland but also inhibits the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. In this study, we examined how GnIH receptors were regulated in pituitary gonadotroph cells and GnRH-producing neurons in the hypothalamus. In the mouse pituitary gonadotroph cell line LβT2, GnRH increased expression of the GnIH receptor, G-protein coupled receptor 74 (GPR74). GnRH also stimulated the expression of GPR74 and GPR147 in primary cultures of rat anterior pituitary cells. In addition, when GnRH was administered to LβT2 cells in a pulsatile manner, low frequency GnRH pulse stimulation stimulated GPR74 and GPR147 expression more than did high frequency GnRH pulses. In the mouse hypothalamic GnRH-producing cell line GT1-7, hypothalamic kisspeptin did not significantly increase the expression of GnIH receptors. However, the intermittent administration of kisspeptin to GT1-7 cells significantly increased GPR74 and GPR147 mRNA expression. The overexpression of either constitutively active MEK kinase (MEKK) or protein kinase A (PKA) in LβT2 cells increased the expression of GPR74 mRNA. Conversely, in GT1-7 cells, although the overexpression of either MEKK or PKA failed to stimulate GnIH receptor expression, the combined overexpression of both kinases together increased GPR74 and GPR147 mRNA levels. Our current observations suggest that two central controllers of reproductive function, GnRH and kisspeptin, stimulate the expression of GnIH receptors in pituitary gonadotroph cells and hypothalamic GnRH neurons.

Cellular Sensitivity to Ferrotoxicity is Associated with Specific Clinical Disorders

Copyright: © 2014 Weinberg ED. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. During the past five decades, scores of medical studies have described the association of ferrotoxicity with development of a great variety of diseases [1]. Increasingly, specific types of body cells are being reported to be injured or killed by low concentrations of iron. In the initial account, cultures of anterior pituitary cells were observed to be killed by 2 μM irons; in this system, hepatocytes remained healthy in 10-100 μM iron [2]. Thus not surprisingly, thalassemic-major children who load iron early in life are deprived of growth hormone. Persons who absorb excessive iron in late teens or early adulthood can suffer from low levels of gonadotrophic hormones. Subsequently, osteoblasts have been recognized to be unusually sensitive to low concentrations of iron [3,4]. In contrast, osteoclasts (cells of macrophage origin) are highly resistant to iron. Thus in iron loaded persons, bone rebuilding by osteoblasts is surpassed by osteoclast destruction of bone. Accordingly, osteoporosis is a common disorder in patients who load iron for genetic, environmental or behavioral reasons [5].

Neuroprotective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the secretin/glucagon/vasoactive intestinal peptide (VIP) super family. PACAP was first isolated from an ovine hypothalamus and named because it stimulated cAMP production in the rat pituitary cell culture. PACAP is widely expressed in the central and peripheral nervous systems. PACAP plays the roles of neurotransmitter, neuromodulator and neurotrophic factors via three Gprotein binding receptors, PAC1, VAPC1 and VPAC2. A number of recent studies have discovered the neuroprotective functions of PACAP in both in vitro and in vivo models. PACAP protects the neurons from death through both direct and indirect ways. PACAP inhibits caspase-3 through cAMP-PKA or MAP kinase-signaling pathways. Moreover, PACAP can stimulate astrocytes to release neuroprotective factors, such as interleukin-6 (IL-6). The present review will briefly summarize the recent studies and provide information for the future use in the clinic.

Interaction of Gonadal Steroids and Gonadotropin-Releasing Hormone on Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP Receptor Expression in Cultured Rat Anterior Pituitary Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are expressed in the hypothalamus, the gonadotrope cells of the anterior pituitary gland, and the gonads, forming an autocrine-paracrine system in these tissues. Within the pituitary, PACAP functions either alone or synergistically with gonadotropin-releasing hormone (GnRH) to stimulate gonadotropin gene expression and secretion. Our goal was to define the hormonal regulation of pituitary PACAP and PACAP receptor (PAC1) gene expression by dihydrotestosterone (DHT), estradiol, and progesterone alone or in conjunction with GnRH. Treatment of adult male rat pituitary cell cultures with DHT or progesterone augmented GnRH-mediated increase in PACAP messenger RNA (mRNA) levels, but neither had an effect when present alone. Conversely, estradiol treatment blunted PACAP gene expression but did not alter GnRH effects on PACAP expression. Expression of PACAP receptor mRNA was decreased by GnRH treatment, minimally increased by DHT treatment, but not altered by the addition of estradiol or progesterone. DHT and GnRH together blunted PACAP receptor gene expression. Taken together, these results suggest that the activity of the intrapituitary PACAP-PAC1 system is regulated via the complex interaction of gonadal steroids and hypothalamic GnRH.

Characterization of sea bass FSHβ 5′ flanking region: transcriptional control by 17β-estradiol

The sea bass follicle-stimulating hormone 5' flanking region (sbFSHβ 5' FR) was cloned and characterized in order to study the molecular mechanisms underlying transcriptional regulation of the sbFSHβ gene. Analysis of the ~3.5kb of this region revealed the presence of several putative cis-acting elements, including steroid hormone response elements, cAMP response elements, pituitary-specific transcription factor response elements, activator protein-1 response elements and TATA sequence. Deleted constructs containing ~3.5kb of the sbFSHβ 5' FR fused to a luciferase reporter gene were transiently transfected into human embryonic kidney (HEK 293) and mouse mature gonadotrope (LβT2) cell lines. The sbFSHβ 5' FR was efficiently expressed under basal conditions in LβT2 but not in HEK 293, pointing to both positive and negative regulatory elements. In order to elucidate the estrogen-mediated sbFSHβ transcriptional activity, in vitro treatments with 17β-estradiol were carried out on primary cultures of pituitary cells and LβT2 cells transiently expressing luciferase under the control of sbFSHβ 5' FR. Overall, these results demonstrate that 17β-estradiol inhibits sbFSHβ gene expression directly at the level of the pituitary. However, it was also shown that estrogen did not induce changes of the sbFSH promoter-directed luciferase activity, suggesting that sbFSHβ 5'FR (~3.5kb) activity is cell type dependent and its estrogen regulation could require cis-acting elements located upstream of the promoter region, which is characterized in this article.

Evidence for an internal and functional circadian clock in rat pituitary cells

In primary cultures of rat pituitary cells and in a pituitary sommatolactotroph cell line (GH4C1), endogenous core-clock- as well as hormone-genes such as prolactin displayed a rhythmic expression pattern, fitted by a sinusoidal equation in which the period value was close to the circadian one. This is consistent with the presence of a functional circadian oscillator in pituitary cells whose importance was ascertained in GH4C1 cell lines stably expressing a dominant negative mutant of BMAL1. In these cells, both endogenous core-clock- and prolactin-genes no more displayed a circadian pattern. Some genes we recently identified as mouse pituitary BMAL1-regulated genes in a DNA-microarray study, lost their circadian pattern in these cells, suggesting that BMAL1 controlled these genes locally in the pituitary. The intra-pituitary circadian oscillator could then play a role in the physiology of the gland that would not be seen anymore as a structure only driven by hypothalamic rhythmic control.

Cadmium mimics estrogen-driven cell proliferation and prolactin secretion from anterior pituitary cells.

Cadmium (Cd) is a heavy metal of considerable occupational and environmental concern affecting wildlife and human health. Recent studies indicate that Cd, like other heavy metals, can mimic effects of 17β-estradiol (E2) involving E2 receptor (ER) activation. Lactotrophs, the most abundant cell type in anterior pituitary gland, are the main target of E2, which stimulates cell proliferation and increases prolactin secretion through ERα. The aim of this work was to examine whether Cd at nanomolar concentrations can induce cell proliferation and prolactin release in anterior pituitary cells in culture and whether these effects are mediated through ERs. Here we show that 10 nM Cd was able to stimulate lactotroph proliferation in anterior pituitary cell cultures from female Wistar rats and also in GH3 lactosomatotroph cell line. Proliferation of somatotrophs and gonadotrophs were not affected by Cd exposure. Cd promoted cell cycle progression by increasing cyclins D1, D3 and c-fos expression. Cd enhanced prolactin synthesis and secretion. Cd E2-like effects were blocked by the pure ERs antagonist ICI 182,780 supporting that Cd acts through ERs. Further, both Cd and E2 augmented full-length ERαexpression and its 46 kDa-splicing variant. In addition, when co-incubated Cd was shown to interact with E2 by inducing ERα mRNA expression which indicates an additive effect between them. This study shows for the first time that Cd at nanomolar concentration displays xenoestrogenic activities by inducing cell growth and stimulating prolactin secretion from anterior pituitary cells in an ERs-dependent manner. Cd acting as a potent xenoestrogen can play a key role in the aetiology of different pathologies of the anterior pituitary and in estrogen-responsive tissues which represent considerable risk to human health.

Differentiation between genomic and non-genomic feedback controls yields an HPA axis model featuring Hypercortisolism as an irreversible bistable switch

BackgroundThe hypothalamic-pituitary-adrenal axis (HPA axis) is a major part of the neuroendocrine system responsible for the regulation of the response to physical or mental stress and for the control of the synthesis of the stress hormone cortisol. Dysfunctions of the HPA axis characterized by either low (hypocortisolism) or increased (hypercortisolism) cortisol levels are implicated in various pathological conditions. Their understanding and therapeutic correction may be supported by mathematical modeling and simulation of the HPA axis.MethodsMass action and Michaelis Menten enzyme kinetics were used to provide a mechanistic description of the feedback mechanisms within the pituitary gland cells by which cortisol inhibits its own production. A separation of the nucleus from the cytoplasm by compartments enabled a differentiation between slow genomic and fast non-genomic processes. The model in parts was trained against time resolved ACTH stress response data from an in vitro cell culture of murine AtT-20 pituitary tumor cells and analyzed by bifurcation discovery tools.ResultsA recently found pituitary gland cell membrane receptor that mediates rapid non-genomic actions of glucocorticoids has been incorporated into our model of the HPA axis. As a consequence of the distinction between genomic and non-genomic feedback processes our model possesses an extended dynamic repertoire in comparison to existing HPA models. In particular, our model exhibits limit cycle oscillations and bistable behavior associated to hypocortisolism but also features a (second) bistable switch which captures irreversible transitions in hypercortisolism to elevated cortisol levels.ConclusionsModel predictive control and inverse bifurcation analysis have been previously applied in the simulation-based design of therapeutic strategies for the correction of hypocortisolism. Given the HPA model extension presented in this paper, these techniques may also be used in the study of hypercortisolism. As an example, we show how sparsity enforcing penalization may suggest network interventions that allow the return from elevated cortisol levels back to nominal ones.

A rapid screening test for endocrine disrupting chemicals using primary cell culture of the marine medaka

While endocrine disrupting chemicals (EDCs) pose a significant threat to wildlife worldwide, their diverse chemical structures present a major challenge to their detection, particularly since they are present at very low concentrations in the environment. We here report the development of an in vitro system for rapid screening of EDCs, using primary cell cultures (pituitary, ovarian follicular and testicular cells) of the marine medaka (Oryzias melastigma). Pituitary, testis and ovary cell cultures were developed and challenged by environmentally relevant concentrations of three well known EDCs (viz. estradiol, 2,2′,4,4′-tetrabromodiphenyl ether, and 4-n-nonylphenol) as well as hypoxia (which has been shown to be a potent endocrine disruptor). In general, the mRNA expression levels of gonadotropins, their receptors and steroidogenic enzymes exhibited dose response relationships to the four endocrine disruptors in different tissues. The sensitivity and responses were also comparable to in vivo responses of whole fish and in vitro responses of the H295R human adrenocortical cell line. Our results suggest that the use of marine medaka primary cultured cells can serve as a cost effective tool for rapid screening of EDCs in the marine environment, and at the same time, sheds light on the underlying mechanisms of EDCs by deciphering their specific target sites along the hypothalamus-pituitary-gonad axis of vertebrates.

The Role of Brain Peptides in the Reproduction of Blue Gourami Males (Trichogaster trichopterus)

In all vertebrates, reproduction and growth are closely linked and both are controlled by complex hormonal interactions at the brain-pituitary level. In this study, we focused on the reciprocal interactions between brain peptides that regulate growth and reproductive functions in a teleostei fish (blue gourami Trichogaster trichopterus). An increase in gonadotropin-releasing hormone 1 (GnRH1) gene expression was detected during ontogeny, and this peptide increased growth hormone (GH) and β follicle-stimulating hormone (βFSH) gene expression in pituitary cell culture. However, although no change in gonadotropin-releasing hormone 2 (GnRH2) gene expression during the reproductive cycle or sexual behavior was detected, a stimulatory effect of this peptide on β gonadotropins (βGtH) gene expression was observed. In addition, pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38) inhibited GnRH-analog-induced βFSH gene expression, and co-treatment of cells with GnRH-analog and PACAP-38 inhibited GnRH-analog-stimulatory and PACAP-38-inhibitory effects on GH gene expression. These findings together with previous studies were used to create a model summarizing the mechanism of brain peptides (GnRH, PACAP and its related peptide) and the relationship to reproduction and growth through pituitary hormone gene expression during ontogenesis and reproductive stages in blue gourami.

Differential modulation of ghrelin-induced GH and LH release by PACAP and dopamine in goldfish pituitary cells

Ghrelin (GRLN) participates in multiple physiological processes, including the regulation of growth hormone (GH) and luteinizing hormone (LH) release. In the goldfish, neuroendocrine control of GH and LH release are multifactorial. In this system, pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated GH and LH secretion, as well as dopamine (DA)-induced GH release, are mediated by protein kinase A (PKA)-dependent, but protein kinase C (PKC)-independent, mechanisms. In addition, DA inhibits LH secretion by actions at sites along both PKA and PKC signaling pathways. Recently, goldfish GRLN (gGRLN19) has been shown to induce GH release via PKC, and LH secretion via both PKC and PKA. To further understand the neuroendocrine regulation of goldfish GH and LH release, we examined the effects of DA and PACAP on gGRLN19 actions in primary cultures of goldfish pituitary cells in perifusion and in Ca2+-imaging experiments. Consistent with their known intracellular signaling mechanisms in gonadotrophs, DA inhibited gGRLN19-induced LH release while cotreatment of PACAP and gGRLN19 did not produce additive LH responses. When applied prior to gGRLN19, PACAP potentiated gGRLN19-induced GH release and Ca2+ signals within somatotrophs. In contrast, neither prior treatment with DA followed by gGRLN19 nor pretreatment with gGRLN19 prior to PACAP produced an enhanced GH release response. These observations suggest that PKA activators positively modulate gGRLN19 actions on goldfish somatotrophs in a ligand- and treatment order-specific manner. Results add to our understanding of the complexity of neuroendocrine control of GH and LH release at the pituitary cell level, and our understanding of GRLN action.

Kisspeptin Regulates Gonadotropin Genes via Immediate Early Gene Induction in Pituitary Gonadotropes

Kisspeptin signaling through its receptor, Kiss1R, is crucial for many reproductive functions including puberty, sex steroid feedback, and overall fertility. Although the importance of Kiss1R in the brain is firmly established, its role in regulating reproduction at the level of the pituitary is not well understood. This study presents molecular analysis of the role of kisspeptin and Kiss1R signaling in the transcriptional regulation of the gonadotropin gene β-subunits, LHβ and FSHβ, using LβT2 gonadotrope cells and murine primary pituitary cells. We show that kisspeptin induces LHβ and FSHβ gene expression, and this induction is protein kinase C dependent and mediated by the immediate early genes, early growth response factor 1 and cFos, respectively. Additionally, kisspeptin induces transcription of the early growth response factor 1 and cFos promoters in LβT2 cells. Kisspeptin also increases gonadotropin gene expression in mouse primary pituitary cells in culture. Furthermore, we find that Kiss1r expression is enhanced in the pituitary of female mice during the estradiol-induced LH surge, a critical component of the reproductive cycle. Overall, our findings indicate that kisspeptin regulates gonadotropin gene expression through the activation of Kiss1R signaling through protein kinase C, inducing immediate early genes in vitro, and responds to physiologically relevant cues in vivo, suggesting that kisspeptin affects pituitary gene expression to regulate reproductive function.

Endogenous Somatostatin Is Critical in Regulating the Acute Effects of l-Arginine on Growth Hormone and Insulin Release in Mice

l-arginine (l-Arg) rapidly stimulates GH and insulin release in vivo. It has been hypothesized that l-Arg stimulates GH release by lowering hypothalamic somatostatin (SST) tone. l-Arg may also act directly at the pituitary to stimulate GH release. Moreover, l-Arg has a direct stimulatory effect on β-cells, which is thought to be blunted by the release of SST from pancreatic δ-cells. To confirm the role of endogenous SST on l-Arg-induced GH and insulin release, wild-type (WT) and SST-knockout (SST-KO) mice were injected with l-Arg (ip; 0.8 g/kg), and pre-/post-injection GH, insulin, and glucose levels were measured. In WT mice, l-Arg evoked a 6-fold increase in circulating GH. However, there was only a modest increase in GH levels in WT pituitary cell cultures treated with l-Arg. In contrast, l-Arg failed to increase GH in SST-KO beyond their already elevated levels. These results further support the hypothesis that the primary mechanism by which l-Arg acutely increases GH in vivo is by lowering hypothalamic SST input to the pituitary and not via direct pituitary effects. Additionally, l-Arg induced a clear first-phase insulin secretion in WT mice, but not in SST-KO. However, SST-KO, but not WT mice, displayed a robust and sustained second-phase insulin release. These results further support a role for endogenous SST in regulating l-Arg-mediated insulin release.

Insulin and IGF-I Inhibit GH Synthesis and Release in Vitro and in Vivo by Separate Mechanisms

IGF-I is considered a primary inhibitor of GH secretion. Insulin may also play an important role in regulating GH levels because insulin, like IGF-I, can suppress GH synthesis and release in primary pituitary cell cultures and insulin is negatively correlated with GH levels in vivo. However, understanding the relative contribution insulin and IGF-I exert on controlling GH secretion has been hampered by the fact that circulating insulin and IGF-I are regulated in parallel and insulin (INSR) and IGF-I (IGFIR) receptors are structurally/functionally related and ubiquitously expressed. To evaluate the separate roles of insulin and IGF-I in directly regulating GH secretion, we used the Cre/loxP system to knock down the INSR and IGFIR in primary mouse pituitary cell cultures and found insulin-mediated suppression of GH is independent of the IGFIR. In addition, pharmacological blockade of intracellular signals in both mouse and baboon cultures revealed insulin requires different pathways from IGF-I to exert a maximal inhibitory effect on GH expression/release. In vivo, somatotrope-specific knockout of INSR (SIRKO) or IGFIR (SIGFRKO) increased GH levels. However, comparison of the pattern of GH release, GH expression, somatotrope morphometry, and pituitary explant sensitivity to acute GHRH challenge in lean SIRKO and SIGFRKO mice strongly suggests the primary role of insulin in vivo is to suppress GH release, whereas IGF-I serves to regulate GH synthesis. Finally, SIRKO and/or SIGFRKO could not prevent high-fat, diet-induced suppression of pituitary GH expression, indicating other factors/tissues are involved in the decline of GH observed with weight gain.