Diurnal rhythms in hypothalamic/pituitary AVT synthesis and secretion in rainbow trout: evidence for a circadian regulation

Arginine vasotocin (AVT) and isotocin (IT) are two neurohypophysial peptide hormones for which a role in adaptation to environmental changes has been suggested in fish. In teleosts, there are only a few available studies about circadian changes of AVT and IT levels, and a role of those peptides in the circadian system has been mainly suggested on the basis of the role of the homologous hormone AVP in mammals. Herein, we evaluated the diurnal rhythms in plasma AVT, pituitary AVT and IT content and the hypothalamic pro-vasotocin (pro-VT) expression in rainbow trout kept under a natural photoperiod, as well as their persistence in constant darkness as a tool for defining circadian dependence. Trout kept under a natural light cycle showed clear diurnal rhythms in both circulating and pituitary AVT levels with peak values around the last hours of the light phase. Hypothalamic pro-VT mRNA was also rhythmically expressed with similar peak characteristics. These rhythms persisted in fish kept under constant darkness for nearly two consecutive days, although peaks were progressively attenuated and phase-advanced. An IT rhythm was also found in pituitary of the trout maintained under a natural photoperiod, but not in those kept under continuous darkness. These results suggest that rhythms of hypothalamic AVT synthesis might be regulated by endogenous circadian mechanisms, and these rhythms contribute to maintain a similar fluctuation in pituitary AVT secretion into the blood. A potential role for AVT in the circadian and seasonal time-keeping system of teleost fish, either as a component of the neural machinery that participates in the adaptation to cyclic environmental changes, or as a circadian/seasonal output signal, is also discussed.     

Chicken growth hormone: further characterization and ontogenic changes of an N-glycosylated isoform in the anterior pituitary gland

Glycosylation is one of the post-translational modifications that growth hormone (GH) can undergo. This has been reported for human, rat, mouse, pig, chicken and buffalo GH. The nature and significance of GH glycosylation remains to be elucidated. This present study further characterizes glycosylated chicken GH (G-cGH) and examines changes in the pituitary concentration of G-cGH during embryonic development and post hatching growth. G-cGH was purified from chicken pituitaries by affinity chromatography (Concanavalin A-Sepharose and monoclonal antibody bound to Sepharose). Immunoreactive G-cGH has a MW of 26 kDa or 29 kDa as determined by SDS-PAGE, respectively, under non-reducing and reducing conditions. Evidence that it is N-glycosylated comes from its susceptibility to peptide N-glycosidase F, and its resistance to O-glycosidase. Based on the ability of G-cGH to bind Concanavalin A or wheat germ agglutinin but not other lectins and its susceptibility to peptide N-glycosidase F, a hybrid or biantennary type glycopeptide (GlcNac2, Man) structure is proposed. Some G-cGH can be observed in the pituitary at most ages examined (from 15-day embryo to adult). Moreover, electron microscopy revealed the presence of both immuno-reactive GH and Concanavalin A-reactive sites in the same secretory granules in the somatotrope. There were marked changes in the level and relative proportion of G-cGH in the pituitary gland during development and growth, the proportion of G-cGH rising during late embryonic development (e.g., between 15 and 18 days of development) and with further increases between 9 weeks and 15 weeks old. G-cGH was able to bind to chicken liver membrane preparations with less affinity than non-glycosylated monomer; on the other hand, however, G-cGH stimulated cell proliferation on Nb2 lymphoma bioassay whereas the non-glycosylated monomer was uncapable to do it.     

Melatonin suppresses cyclosporine A‐induced autophagy in rat pituitary GH3 cells

Abstract: Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the induction of chronic nephrotoxicity including endoplasmic reticulum (ER) stress in tubular cells. Recently, it was reported that autophagy is induced by ER stress and serves to alleviate the associated deleterious effects. In the current study, CsA treatment (0–100 μm ) decreased cell survival of rat pituitary GH3 cells in a dose‐dependent manner. At concentrations ranging from 1.0 to 10 μm , CsA induced a dose‐dependent increase in the expression of microtubule‐associated protein 1 light chain 3 (LC3)‐I and LC3‐II. Cells treated with 2.5 μm CsA exhibited cytoplasmic vacuolation, indicating that CsA induces autophagy in rat pituitary GH3 cells. In the presence of 1.0–10 μm CsA, the expression of catalase decreased while that of the ER stress markers, ER luminal binding protein (BiP) and inositol‐requiring enzyme 1 alpha (IRE1α), increased as compared those levels in untreated cells. These results suggested that CsA‐induced autophagy is dependent on ER stress. To determine whether melatonin would protect cells against CsA‐induced autophagy, we treated rat pituitary GH3 cells with melatonin in the presence of CsA. Melatonin treatment (100 and 200 μm ) suppressed autophagy induced by 2.5 and 5 μm CsA. Furthermore, co‐treatment with 100 μm melatonin inhibited LC3‐II expression, and increased catalase and phosphorylated p‐ERK levels in the presence of 2.5 and 5 μm CsA. BiP and IRE1α expression in melatonin‐co‐treated cells was superior to that in cells treated with 2.5 and 5 μm CsA alone. Thus, melatonin suppresses CsA‐mediated autophagy in rat pituitary GH3 cells.     

The regulation of central and peripheral circadian clocks in humans

Many circadian rhythms are controlled by the central clock of the suprachiasmatic nucleus of the hypothalamus, as well as clocks located in other brain regions and most peripheral tissues. These central and peripheral clocks are based on clock genes and their protein products. In recent years, the expression of clock genes has started to be investigated in human samples, primarily white blood cells, but also skin, oral mucosa, colon cells, adipose tissue as well as post-mortem brain tissue. The expression of clock genes in those peripheral tissues offers a way to monitor human peripheral clocks and to compare their function and regulation with those of the central clock, which is followed by markers such as melatonin, cortisol and core body temperature. We have recently used such an approach to compare central and peripheral rhythms in subjects under different lighting conditions. In particular, we have monitored the entrainment of the clock of blood cells in subjects undergoing a simulated night shift protocol with bright light treatment, known to efficiently reset the central clock. This line of research will be helpful for learning more about the human circadian system and to find ways to alleviate health problems of shift workers, and other populations experiencing altered circadian rhythms.     

Modulation of VEGF/Flk-1 receptor expression in the rat pituitary GH3 cell line by growth factors

Both vascular endothelial growth factor (VEGF) and its receptor Flk-1 are expressed in normal pituitary cells and in the prolactin- and growth hormone-producing GH3 cell line of the rat, thus suggesting autocrine/paracrine function. Regulation of the Flk-1 receptor system in pituitary cells is poorly understood, but evidence suggests that up-regulated growth factors play a role in its expression and activation. To study the role of growth factors in this process, we examined changes in VEGF and Flk-1 expression in GH3 cells following varied exposure to βFGF, EGF, and TGFβ1. Immunofluorescence labelling and laser scanning cytometry were used to measure changes in VEGF and Flk-1 expression. Results showed that βFGF, EGF and TGFβ up-regulated the VEGF/FLK-1 receptor system. Distinct patterns of activation were detected. At 2 hours, EGF and TGFβ caused no significant changes in VEGF and Flk-1 expression; however, βFGF up-regulated VEGF expression in 99% of cells but only induced modest changes in Flk-1 overexpression. A similar percentage of cells overexpressed VEGF after 24-hour incubation with βFGF, but more prominent Flk-1 overexpression was detected. At 24 hours, EGF and TGFβ1 induced a significant increase in both VEGF and Flk-1 expression. In summary, our findings show that VEGF/Flk-1 expression in pituitary cells may be altered by different growth factors. This may affect angiogenesis and the progression of pituitary tumors.     

Peripheral circadian clock for the cuticle deposition rhythm in Drosophila melanogaster

Insect endocuticle thickens after adult emergence by daily alternating deposition of two chitin layers with different orientation. Although the cuticle deposition rhythm is known to be controlled by a circadian clock in many insects, the site of the driving clock, the photoreceptor for entrainment, and the oscillatory mechanism remain elusive. Here, we show that the cuticle deposition rhythm is regulated by a peripheral oscillator in the epidermis in Drosophila melanogaster. Free-running and entrainment experiments in vitro reveal that the oscillator for the cuticle deposition rhythm is independent of the central clock in the brain driving the locomotor rhythms. The cuticle deposition rhythm is absent in null and dominant-negative mutants of clock genes (i.e., period, timeless, cycle, and Clock), indicating that this oscillator is composed of the same clock genes as the central clock. Entrainment experiments with monochromatic light-dark cycles and cry(b) flies reveal that a blue light-absorbing photoreceptor, cryptochrome (CRY), acts as a photoreceptor pigment for the entrainment of the cuticle deposition rhythm. Unlike other peripheral rhythms in D. melanogaster, the cuticle deposition rhythm persisted in cry(b) and cry(OUT) mutant flies, indicating that CRY does not play a core role in the rhythm generation in the epidermal oscillator.     

Acute ethanol disrupts photic and serotonergic circadian clock phase-resetting in the mouse

Background: Alcohol dependence is associated with impaired circadian rhythms and sleep. Ethanol administration disrupts circadian clock phase‐resetting, suggesting a mode for the disruptive effect of alcohol dependence on the circadian timing system. In this study, we extend previous work in C57BL/6J mice to: (i) characterize the suprachiasmatic nucleus (SCN) pharmacokinetics of acute systemic ethanol administration, (ii) explore the effects of acute ethanol on photic and nonphotic phase‐resetting, and (iii) determine if the SCN is a direct target for photic effects. Methods: First, microdialysis was used to characterize the pharmacokinetics of acute intraperitoneal (i.p.) injections of 3 doses of ethanol (0.5, 1.0, and 2.0 g/kg) in the mouse SCN circadian clock. Second, the effects of acute i.p. ethanol administration on photic phase delays and serotonergic ([+]8‐OH‐DPAT‐induced) phase advances of the circadian activity rhythm were assessed. Third, the effects of reverse‐microdialysis ethanol perfusion of the SCN on photic phase‐resetting were characterized. Results: Peak ethanol levels from the 3 doses of ethanol in the SCN occurred within 20 to 40 minutes postinjection with half‐lives for clearance ranging from 0.6 to 1.8 hours. Systemic ethanol treatment dose‐dependently attenuated photic and serotonergic phase‐resetting. This treatment also did not affect basal SCN neuronal activity as assessed by Fos expression. Intra‐SCN perfusion with ethanol markedly reduced photic phase delays. Conclusions: These results confirm that acute ethanol attenuates photic phase‐delay shifts and serotonergic phase‐advance shifts in the mouse. This dual effect could disrupt photic and nonphotic entrainment mechanisms governing circadian clock timing. It is also significant that the SCN clock is a direct target for disruptive effects of ethanol on photic shifting. Such actions by ethanol could underlie the disruptive effects of alcohol abuse on behavioral, physiological, and endocrine rhythms associated with alcoholism.     

Circadian rhythms in the development of obesity: potential role for the circadian clock within the adipocyte

Obesity is one of the most profound public health problems today, and simplistic explanations based on excessive nutritional consumption or lack of physical activity are inadequate to account for this dramatic and literal growth in our world population. Recent reports have suggested that disruptions in sleep patterns, often linked to our '24-h' lifestyle, are associated with increased body fat and altered metabolism, although the cause-effect relationship for these associations has yet to be elucidated. Abnormal sleep/wake patterns likely alter intracellular circadian clocks, which are molecular mechanisms that enable the cell/tissue/organism to anticipate diurnal variations in its environment. The environment may include circulating levels of nutrients (e.g. glucose, fatty acids and triglycerides) and various hormones (e.g. insulin, glucocorticoids). As such, alterations in this molecular mechanism, in particular within the adipocyte, likely induce metabolic changes that may potentiate disrupted metabolism, adipose accumulation and/or obesity. Although diurnal variations in adipokines and adipose tissue metabolism have been observed, little is known regarding the molecular mechanisms that influence these events.     

Dopamine receptor D2S gene transfer improves the sensitivity of GH3 rat pituitary adenoma cells to bromocriptine

Bromocriptine, a dopamine agonist (DA), has been used in the treatment of prolactinomas. Recent studies have indicated that dopamine 2 receptor short isoform (D2S) may play an important role in suppressing PRL synthesis and prolactinoma cell growth under DA treatment. In the current study, we investigated the role of D2S in the therapeutic action of bromocriptine in GH3 using both in vitro and in vivo approaches. Infection of adenovirus-D2S increased D2S expression in GH3 cells (P < 0.05). D2S expression significantly decreased the GH3 cell viability subjected to bromocriptine treatment in vitro (P < 0.05). In nude mice, adenovirus-D2S transfection sensitized GH3 xenograft to bromocriptine treatment evidenced by the significant inhibition of D2S expressed tumor growth as compared with vector control. Furthermore, decrease of Bcl-2 expression, increase of Bax, and active Caspase-3 were found in D2S expressed GH3 xenograft subjected to bromocriptine treatment. In summary, our study indicates that D2S expression plays a critical role in the therapeutic action of bromocriptine in pituitary adenomas and that adenovirus-mediated D2S gene transfer combined with bromocriptine may provide a novel treatment for DA-resistant prolactinomas.     

Evidence for an unidentified factor from the pituitary gland which affects the steroid metabolism in isolated hepatocytes and hepatoma cells of the rat

The effects of various extracts obtained from normal rat pituitary tissue (of female origin) and pituitary tumor tissue on the metabolism of 4-androstene-3,17-dione in isolated rat hepatocytes and cultured hepatoma (HTC) cells were investigated. Extracts that increased the 5-reductase/16-hydroxylase ratio in isolated hepatocytes also increased the 5-reductase activity or the 5-reductase/17-hydroxysteroid dehydrogenase ratio in HTC cells. These results indicate that both cell culture methods are suitable for use as in vitro assays for the pituitary principle which affects hepatic steroid metabolism in vivo.

Using the above cell culture technique it has been possible to show that ‘feminizing factor’ activity is located in granules (densities 1.13 and 1.17g/cm³, respectively) in the female rat pituitary and in extracts from the cloned pituitary tumor (C₈11RAP). ‘Feminizing factor’ activity could not be duplicated by single standard anterior or posterior gland hormone preparations over a wide range of concentrations or combinations of such preparations. These results further strengthen the hypothesis put forward in earlier publications that an as yet unidentified pituitary principle may be involved in the control of hepatic steroid metabolism in the rat.     

垂体柄阻断综合征的病因分析及诊疗进展

垂体柄阻断综合征(PSIS)的病因和发病机制目前尚不明确.近来研究表明,有遗传因素参与PSIS的发生、发展,尤以HESX1、LHX4基因缺陷最为密切.PSIS的临床表现复杂多样,随不同年龄及不同垂体激素缺乏而表现各异.早期诊治对患者激素缺乏症状的改善至关重要,MRI是唯一能明确此病诊断的影像学方法,胰岛素样生长因子及其结合蛋白3可与MRI联合提高对PSIS的诊断率.     

The permeant molecule urea stimulates prolactin secretion in GH4C1 cells by inducing Ca2+ influx through dihydropyridine-sensitive Ca2+ channels

Isotonic urea in medium with a normal 1.2 mM Ca²⁺ concentration induced a striking rise in both cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and prolactin (PRL) secretion, each of whose peaks were proportional to the concentration of urea between 5 and 120 mM. There was a significant linear relationship between the peaks of induced [Ca²⁺]_i and PRL secretion (r = 0.99, P < 0.001). The increase in both [Ca²⁺]_i and PRL secretion was completely abolished by removal of medium Ca²⁺ or by 2 μM nifedipine. Hypertonie urea was ineffective in inducing either an increase in [Ca²⁺]_i or PRL secretion. These data support the hypothesis that plasma membrane expansion is a potent non-toxic inducer of hormone secretion and that in GH₄C₁ cells an increase in [Ca²⁺]_i produced by enhanced influx of extracellular Ca²⁺ through dihydropyridine-sensitive Ca²⁺ channels plays an important role in this phenomenon.     

Evidence for functional communication between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary cell aggregates

Dispersed anterior pituitary cells from adult female rats were separated by gradient sedimentation at unit gravity. The small-sized cell population on top of the gradient consisted of 65.6 +/- (SE) 4.2% (n = 8) cells immunoreactive to antiserum against S-100 protein, a marker of folliculo-stellate (FS) cells in rat pituitary. The corresponding fraction derived from adult male or immature female rats were also enriched in S-100 positive cells but to a lower extent. Only small numbers of S-100 positive cells were found in medium- and large-sized cell populations. Coaggregating the S-100 cell-enriched populations from adult females with other pituitary cell populations resulted in a clear-cut inhibition of the GH response to rat GH-releasing factor and beta-adrenergic agents, of the PRL response to TRH and angiotensin II (AII) and the LH response to LHRH. The magnitude of inhibition increased with the number of FS cells put into the coaggregates. In perifused aggregates prepared from different gradient fractions from immature females, there was a negative correlation between the occurrence of FS cells and the magnitude of the PRL response to AII. The low responsiveness to AII in FS cell enriched aggregates was not abolished when these aggregates were redissociated into single cells. It is suggested that FS cells constitute an intercellular messenger system for local inhibitory control of pituitary hormone secretion which is not based on direct and intimate contact between the interacting cells.