GT1-7 Cells Research Articles

119-OR: Amplified Glucagon Response to Hypoglycemia following AMP-Activated Protein Kinase (AMPK) Activator R481 Treatment in Healthy Rats

Episodes of insulin-induced hypoglycemia are frequent in type 1 and advanced type 2 diabetes, but pharmaceutical approaches to prevent these are lacking. Cellular stresses such as low blood sugar activate AMPK, which has emerged as a whole body and cellular energy sensor. Direct delivery of AMPK activator to the ventromedial hypothalamus of rodents leads to increased hepatic glucose-production, observed during hyperinsulinemic-hypoglycemic clamp studies, and genetic activation of AMPK in the pancreatic alpha cell increases glucagon release. It may therefore be suitable to target AMPK for hypoglycemia prevention. Here, R481, a novel metformin-like brain permeable AMPK activator, was used to assess the impact of AMPK pathway activation on the counterregulatory response to hypoglycemia. Hypothalamic glucose sensing GT1-7 cells were treated with R481 and activation of AMPK pathway by phosphorylation assessed using Western Blotting. R481 was administered orally to male Sprague Dawley rats prior to insulin-induced hypoglycemia, following which blood glucose and feeding were measured. A separate cohort of rats underwent a hyperinsulinemic-hypoglycemic clamp study where glucose infusion rates and counterregulatory hormones levels were determined. Nanomolar concentrations of R481 increased AMPK pathway phosphorylation in GT1-7 neurons. Administration of R481 (5-20 mg/kg) to rats attenuated insulin-mediated drop in blood glucose during acute insulin-induced hypoglycemia, without altering fast-induced refeeding. R481 decreased the glucose infusion rate during hyperinsulinemic-hypoglycemic clamps, by amplifying plasma glucagon secretion, without altering epinephrine. Peripheral administration of AMPK activator R481 amplified glucagon release to improve counterregulatory response to hypoglycemia in healthy rats. Disclosure A.M.L. Cruz: None. Y. Malekizadeh: None. J.M. Vlachaki Walker: None. S.J. Shaw: Employee; Self; Rigel Pharmaceuticals. K.L. Ellacott: None. C. Beall: None. Funding JDRF; Diabetes UK

Involvement of Kisspeptin in Androgen-Induced Hypothalamic Endoplasmic Reticulum Stress and Its Rescuing Effect in PCOS Rats

Background: Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and Endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. Methods: An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, We detected Kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. Findings: We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly down-regulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1-7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1-7 cells, as well as in the hypothalamus in PCOS rats. Similarly, Kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1-7 cells. Interpretation: Our data has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent. Funding Statement: National Basic Research Program of China, National Natural Science Foundation of China (2012CB944902, 2012CB944703, 2013CB967404, 2013CB967401, 2013CB967403, 81571403, 81471429) and State Key laboratory of Reproductive Medicine (SKLRM-K201805). Declaration of Interests: The authors have no conflicts of interest about this work. Ethics Approval Statement: The study was approved by the Ethics Committee of Tongji University and all patients gave informed consent. All procedures in this study were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong, and the methods were used in accordance with the guidelines of the Chinses University of Hong Kong.

Vasopressin regulates hypothalamic GnRH synthesis: Histomorphological evidence in hypothalamus and biological effects in GT1-7 cells

AimsTo investigate the direct histomorphological clues and observe the biological effects of VP acting on gonadotropin-releasing hormone (GnRH) secretion. Main methodsImmunofluorescence was conducted to investigate the expressions of GnRH and VP in experimental left varicocele (ELV) rats and ELV repair rats. The colocalization of GnRH and VP was observed by electron microscopy immunohistochemistry. The protein-protein interaction between GnRH and VP was tested by co-immunoprecipitation (co-IP) and the proximity ligation assay (PLA). The effects of intracellular and extracellular VP on GnRH and relative transcription factors (Oct-1, Otx2, Pbx1b and DREAM) were respectively evaluated in VP overexpressed and VP treated GT1-7 cells. Key findingsBoth hypothalamic GnRH and VP decreased in ELV rats and recovered by ELV repair. The overlapped immunolocalizations of GnRH and VP mainly distributed in the lateral part of the arcuate nucleus (ArcL) and median eminence (ME) with a Manders' overlap coefficient of 0.743 ± 0.117. Immunoreactive substances of GnRH and VP existed in the same and adjacent terminals. VP overexpression did not cause any significant effects on the expressions of GnRH and Oct-1, as well as GnRH promoter activity. While 50–200 pg/ml VP treatments increased GnRH mRNA levels in a dose- and time-dependent manner in GT1-7 cells. Additionally, 200 pg/ml VP triggered a marked promotion of expressions of GnRH, Oct-1, Oxt2 Pbx1b and DREAM, as well as GnRH promoter activity (P < 0.05). SignificanceThe results reveal the colocalization and interaction of VP and GnRH, which will be conducive to explain the effects and mechanisms of VP acting on reproduction.

MON-031 In Vitro Exposure to Bisphenol A, Benzophenones 2 or 3, Have Different Effects on GnRH Gene Expression and Secretion in Mature and Immature GnRH Neurons

Bisphenol A, (BPA), a component of polycarbonate plastics, and Benzophenones (BPs), UV filters, are endocrine disrupting chemicals (EDC). Previously we showed that the in vitro exposure to BPA decreased Kiss-induced GnRH expression in GN11 cells (Dr. Susan Wray, NIH), immature GnRH neurons (1). In this study, we analyzed the in vitro effects of BPA, BP2 and BP3 (1x10-9M), or medium as control (C) in mature GnRH neurons (GT1-7 cells, donated by Dr Pamela Mellon, UCSD, USA, and isolated hypothalami from adult Balb/c males). Effects of BP2 and BP3 (1x10-9M) were also analyzed in GN11 cells. We exposed the cell lines for 24 h and the hypothalami for 6 h to the EDC. GT1-7 and GN11 cells were further incubated with Kiss (1x10-9M, Phoenix Pharmaceuticals, Inc, CA, USA) for 4 h to analyze gene expression (qPCR) and for 1 h to analyze GnRH secretion (RIA). Hypothalami exposed to BPA showed increased GnRH expression compared to C (Relative expression: C:1.0±0.2; BPA:2.2±0.5; Student T-test: BPA vs C p<0.05, n=9), whereas neither BP2 nor BP3 had any effect (ANOVA: ns). There was no effect on GnRH secretion (ANOVA: ns). GT1-7 cells exposed to C showed Kiss-induced GnRH gene expression, while the exposure to the EDC abolished this response (Relative expression: C-Basal:1.0±0.1, C-Kiss:1.6±0.3, BPA-Basal:1.3±0.2, BPA-Kiss:1.1±0.2, BP2-Basal:1.2±0.1, BP2-Kiss:1.4±0.2, BP3-Basal:1.3±0.3, BP3-Kiss:1.1±0.2; Repeated measures Two-way ANOVA: Interaction p<0.05, n=6). There was no difference in Kiss-stimulated GnRH release in EDC-exposed cells vs C-exposed cells (Repeated measures Two-way ANOVA: Interaction ns; Main Effect: Time of Kiss exposure: p<0.01, n=7). In GN11 cells, exposure to BP2 inhibited Kiss-induced GnRH gene expression, whereas there was no such effect in cells exposed to BP3 (Relative Expression: C-Basal:1.1±0.02, C-Kiss:1.3±0.1, BP2-Basal:1.2±0.1, BP2-Kiss:1.0±0.1, BP3-Basal:1.0±0.1, BP3-Kiss:1.3±0.1; Repeated measures Two-way ANOVA: Interaction p<0.01, n=8). Exposure to BP3 significantly increased GnRH secretion and abolished the response to Kiss, whereas BP2 significantly decreased Kiss-stimulated release [GnRH (pg/ml): C-Basal: 8.5±1.3, C-Kiss: 32.9±8.8, BP2-Basal: 16.5±4.9, BP2-Kiss: 16.9±5.1, BP3-Basal: 18.0±3.2, BP3-Kiss: 20.8±5.6, Repeated measures Two-way ANOVA: Interaction p<0.05; C-Kiss vs C-Basal, BP3-Basal vs C-Basal, BP2-Kiss vs C-Kiss p<0.05, n=9]. Our results show that EDC exposure alters the physiology of mature and immature GnRH neurons, although effects are different in the two cell lines, and they also differ in mature GnRH neurons vs hypothalamus. Further studies are needed to dissect the mechanisms involved. 1- Endoc. Rev. 38(3S), SAT-256, 2017. Nothing to disclose. Funding: CONICET, ANPCyT, UBA, International Society for Neurochemistry, Asoc. ORT Arg., Fund. R. Barón, Fund. Williams.

SUN-468 Sex Differences in Obesity-Mediated Impairment of Reproductive Function

Increasing prevalence in obesity has become a significant public concern. C57BL/6J mice are prone to diet-induced obesity (DIO) when fed high-fat diet (HFD), and develop chronic inflammation and metabolic syndrome, making them a good model to analyze mechanisms whereby obesity elicits pathologies. DIO mice demonstrated profound sex differences in response to HFD with respect to inflammation and hypothalamic function. First, we determined that males are prone to DIO, while females are resistant. Ovariectomized females, on the other hand, are susceptible to DIO, implying protection by ovarian hormones. Males, but not females, exhibit changes in gonadotropin hormone concentration and hypothalamic neuropeptide expression. Surprisingly, ovariectomized females remain resistant to neuroendocrine changes, showing that ovarian hormones are not necessary for protection. Second, obese mice exhibit sex differences in DIO-induced inflammation. Proinflammatory cytokines are increased specifically in males, while anti-inflammatory cytokines are increased in the hypothalami of females but not males, which may provide protection to female mice. Again, females are protected from the increase in inflammatory cytokines regardless of the presence of ovarian hormones. Third, males and females deposit fat differentially in subcutaneous and visceral depots. Obese males exhibit reduced synapses in GnRH neurons and diminished GnRH gene expression, which may stem from obesity-mediated activation of immune cells or from direct effect of proinflammatory cytokines on GnRH gene expression, respectively. Proinflammatory cytokines directly repress GnRH gene expression in GT1-7 cells model, while in vivo, obese males show activation of a cytokine-induced transcription factor in GnRH neurons. Our studies suggest that inflammation-induced hypothalamic changes are potentially responsible for diminished levels of gonadotropin hormones, testosterone, and sperm numbers, which corresponds to the observations in obese humans. Taken together, we propose a potential mechanism of obesity-induced impairment of hypothalamic function, and our data implicate neuro-immune mechanisms underlying sex-specific differences in obesity-induced impairment of the hypothalamic function with potential consequences for reproduction and fertility.

Involvement of SAPK/JNK Signaling Pathway in Copper Enhanced Zinc-Induced Neuronal Cell Death

Zinc (Zn) plays an important role in many organisms in various physiological functions such as cell division, immune mechanisms and protein synthesis. However, excessive Zn release is induced in pathological situations and causes neuronal cell death. Previously, we reported that Cu ions (Cu2+) markedly exacerbates Zn2+-induced neuronal cell death by potentiating oxidative stress and the endoplasmic reticulum stress response. In contrast, the stress-activated protein kinase/c-Jun amino-terminal kinase (SAPK/JNK) signaling pathway is important in neuronal cell death. Thus, in this study, we focused on the SAPK/JNK signaling pathway and examined its involvement in Cu2+/Zn2+-induced neurotoxicity. Initially, we examined expression of factors involved in the SAPK/JNK signaling pathway. Accordingly, we found that phosphorylated (ie, active) forms of SAPK/JNK (p46 and p54) are increased by CuCl2 and ZnCl2 co-treatment in hypothalamic neuronal mouse cells (GT1-7 cells). Downstream factors of SAPK/JNK, phospho-c-Jun, and phospho-activating transcription factor 2 are also induced by CuCl2 and ZnCl2 co-treatment. Moreover, an inhibitor of the SAPK/JNK signaling pathway, SP600125, significantly suppressed neuronal cell death and activation of the SAPK/JNK signaling pathway induced by CuCl2 and ZnCl2 cotreatment. Finally, we examined involvement of oxidative stress in activation of the SAPK/JNK signaling pathway, and found that human serum albumin-thioredoxin fusion protein, an antioxidative protein, suppresses activation of the SAPK/JNK signaling pathway. On the basis of these results, our findings suggest that activation of ZnCl2-dependent SAPK/JNK signaling pathway is important in neuronal cell death, and CuCl2-induced oxidative stress triggers the activation of this pathway.

MiR-505-3p is a repressor of the puberty onset in female mice.

Puberty onset is a complex trait regulated by multiple genetic and environmental factors. In this study, we narrowed a puberty related QTL region down to a 1.7 Mb region on chromosome X in female mice and inferred miR-505-3p as the functional gene. We conducted ectopic expression of miR-505-3p in the hypothalamus of prepubertal female mice through lentivirus-mediated orthotopic injection. The impact of miR-505-3p on female puberty was evaluated by the measurement of pubertal/reproduction events and histological analysis. The results showed that female mice with overexpression of miR-505-3p in the hypothalamus manifested later puberty onset timing both in vaginal opening and ovary maturation, followed by weaker fertility lying in the longer interval time between mating and delivery, higher abortion rate and smaller litter size. We also constructed miR-505-3p knockout mice by CRISPR/Cas9 technology. MiR-505-3p knockout female mice showed earlier vaginal opening timing, higher serum gonadotrophin and higher expression of puberty-related gene in the hypothalamus than their wild type littermates. Srsf1 was proved to be the target gene of miR-505-3p that played the major role in this process. The results of RNA Immunoprecipitation-sequencing showed that SRSF1 (or SF2), the protein product of Srsf1 gene, mainly bound to ribosome protein (RP) mRNAs in GT1-7 cells. The collective evidence implied that miR-505-3p/SRSF1/RP could play a role in the sexual maturation regulation of mammals.

AgRP/NPY Neuron Excitability Is Modulated by Metabotropic Glutamate Receptor 1 During Fasting.

The potential to control feeding behavior via hypothalamic AgRP/NPY neurons has led to many approaches to modulate their excitability—particularly by glutamatergic input. In the present study using NPY-hrGFP reporter mice, we visualize AgRP/NPY neuronal metabotropic glutamate receptor 1 (mGluR1) expression and test the effect of fasting on mGluR1 function. Using the pharmacological agonist dihydroxyphenylglycine (DHPG), we demonstrate the enhanced capacity of mGluR1 to drive firing of AgRP/NPY neurons after overnight fasting, while antagonist 3-MATIDA reduces firing. Further, under synaptic blockade we demonstrate that DHPG acts directly on AgRP/NPY neurons to create a slow inward current. Using an in vitro approach, we show that emulation of intracellular signals associated with fasting by forskolin enhances DHPG induced phosphorylation of extracellularly regulated-signal kinase (1/2) in GT1-7 cell culture. We show in vivo that blocking mGluR1 by antagonist 3-MATIDA lowers fasting induced refeeding. In summary, this study identifies a novel layer of regulation on AgRP/NPY neurons integrated with whole body energy balance.

Antisense techniques provide robust decrease in GnRH receptor expression with minimal cytotoxicity in GT1-7 cells

ABSTRACTThe episodic pattern of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is driven by an integrated network of cells termed the GnRH pulse generator. Cultured and immortalized GnRH neurons also produce a pulsatile pattern of GnRH secretions when grown in the absence of other cell types, suggesting the presence of an intrinsic oscillator mediating GnRH secretion. The mechanisms underlying such pulsatility comprise one of the most tantalizing problems in contemporary neuroendocrinology. In order to study the mechanism by which GnRH is produced in a pulsatile fashion, the autocrine effect of GnRH on GnRH-producing neurons must be eliminated. This may be performed by downregulating the expression of the GnRH receptor. Treatment with three 21-mer exogenous phosphorothioates and transient transfections with an inducible plasmid containing an antisense construct to the GnRH receptor gene decreased GnRH receptor expression further. This resulted in less cytotoxicity compared to inhibition of RNA or protein synthesis with actinomycin D, α-amanitin, puromycin, and cycloheximide. This study shows methods and optimized conditions established for the generation of a stable GT1-7 cell line containing an inducible construct allowing the downregulation of GnRH receptor expression.Abbreviations: ANOVA: analysis of the variance; DMEM: Dulbecco’s modified Eagle’s medium; GnRH: gonadotropin-releasing hormone; RXR: retinoid X receptor

Role of EGFL7/EGFR-signaling pathway in migration and invasion of growth hormone-producing pituitary adenomas.

Currently, the primary therapeutic strategy for most growth hormone-producing pituitary adenomas (GHPA) is surgery. Due to the invasiveness of GHPA, high recurrence has limited the benefit of complete adenoma removal surgery. Epidermal growth factor-like domain 7 (EGFL7) is a secreted factor implicated in tumor angiogenesis, growth, invasiveness and metastasis in GHPA. Herein, we observed that the expression level of EGFL7 and p-EGFR in invasive GHPA was much higher than that of non-invasive GHPA. The overexpression of EGFL7 was positively correlated with activation of EGFR (p-EGFR). Noticeably, EGFL7 knockdown significantly inhibited activation of EGFR signaling cascades, including p-ERGR, p-AKT and p-ERK. Further studies showed that EGFL7 knockdown or pharmacological inhibition of EGFR-pathway, using EGFR inhibitor Tyrphostin AG-1478, significantly suppressed migration and invasion of GH3 and GT1-1 cells. In summary, our findings suggest that EGFL7 is a key factor for regulation of EGFR signaling pathway and plays an important role in migration and invasion of invasive GHPA.

Up-regulation of DUSP5 and DUSP6 by gonadotropin-releasing hormone in cultured hypothalamic neurons, GT1-7 cells.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons) and stimulates anterior pituitary gonadotrophs to synthesize and secrete gonadotropins. In addition to gonadotrophs, GnRH neurons also express GnRH receptors, and the autocrine action of GnRH is reportedly involved in the regulation of functions of GnRH neurons. There is accumulating evidence that extracellular signal-regulated kinase (ERK), one of mitogen-activated protein kinases (MAPKs), is activated by GnRH and involved in various effects of GnRH in GnRH neurons. In the present study, we performed microarray analysis to examine the types of genes whose expression was regulated by GnRH in immortalized mouse GnRH neurons (GT1-7 cells). We found that 257 genes among 55,681 genes examined were up-regulated after 30-min treatment of GT1-7 cells with GnRH. These up-regulated genes included four dual-specificity MAPK phosphatases (DUSPs), DUSP1, DUSP2, DUSP5, and DUSP6. Reverse transcription-polymerase chain reaction analysis confirmed that the mRNA levels of DUSP5 and DUSP6 were robustly increased within 30 min. U0126, an inhibitor of ERK activation, completely inhibited the increases in the mRNA levels of DUSP5 and DUSP6. Immunoblotting analysis revealed that ERK activation peaked at 5 min and declined steeply at 60 min, whereas DUSP5 and DUSP6 proteins were increased from 60 min. It was notable that down-regulation of DUSP6 augmented GnRH-induced ERK activation approximately 1.7-fold at 60 min. These results suggested that the up-regulation of DUSP6 regulates the duration of ERK activation at least in part.

Kisspeptin increases intracellular calcium concentration by protein kinase C-mediated signaling in the primary cultured neurons from rat hippocampus

In addition to the fact that kisspeptin and its receptor GPR54 are well known to be abundantly expressed in the hypothalamus with suggestive roles in the initiation of puberty and similar reproductive system properties, there is also proof showing that kisspeptin might have influences on hippocampal functions. In our previous study, it was shown that kisspeptin increased free intracellular Ca2+ values ([Ca2+]i) through protein kinase C (PKC) activation in GT1-7 cells. For this reason, we examined the influences of kisspeptin on [Ca2+]i in hippocampal neurons to determine if kisspeptin shows its effects on hippocampus through the same mechanism. Hippocampal neurons were excised from the brains of fetuses on 17th embryonic day from maternal rats. The influences of kisspeptin on [Ca2+]i in hippocampal neurons were examined through in vitro calcium imaging system. The responses of [Ca2+]i to kisspeptin were quantified by the changes in 340nm/380nm ratio. Kisspeptin-10 caused [Ca2+]i transients in hippocampal neurons. The change in [Ca2+]i by 100 nM kisspeptin was prevented by pre-treating the cells in PKC inhibitor chelerythrine chloride. According to the results, kisspeptin activates intracellular calcium signaling in hippocampal neurons via the pathway that depends on PKC. The results of this study suggest that kisspeptin may have a role in hippocampal neuron functions.

Attenuation of EGFL7 Expression Inhibits Growth Hormone-Producing Pituitary Adenomas Growth and Invasion.

Invasiveness of growth hormone-producing pituitary adenomas (GHPAs) causes difficulties in safe and complete adenoma removal during surgery and often leads to high recurrence. Epidermal growth factor-like domain 7 (EGFL7) has been shown to be able to promote tumor angiogenesis, growth, invasiveness, and metastasis through the Notch signaling pathway. It was previously demonstrated that EGFL7 was overexpressed in GHPAs. This study reports that EGFL7 and Notch2 (positive correlation with EGFL7) are overexpressed in invasive GHPA. A long-rank test (Kaplan-Meier method) shows that invasive GHPAs with EGFL7 strong expression results in reduced recurrence-free survival. Multivariate Cox regression analysis reveals that weak EGFL7 expression is an independent prognostic factor for recurrence-free survival. In addition, knockdown of EGFL7 expression suppresses proliferation and invasion of GH3 and GT1-1 cells in vitro. Moreover, attenuation of EGFL7 inhibits human GHPA growth in vivo. The data suggest that as a Notch agonist, EGFL7 may potentially be an appropriate novel molecular target for future development of GHPA medical therapy.

Pyruvic acid prevents Cu2+/Zn2+-induced neurotoxicity by suppressing mitochondrial injury

Zinc (Zn) is known as a co-factor for over 300 metalloproteins or enzymes, and has essential roles in many physiological functions. However, excessively high Zn concentrations are induced in pathological conditions such as interruption of blood flow in stroke or transient global ischemia-induced neuronal cell death. Furthermore, we recently found that copper (Cu2+) significantly exacerbates Zn2+ neurotoxicity in mouse hypothalamic neuronal cells, suggesting that Zn2+ interaction with Cu2+ is important for the development of neurological disease. Meanwhile, organic acids such as pyruvic acid and citric acid are reported to prevent neuronal cell death induced by various stresses. Thus, in this study, we focused on organic acids and searched for compounds that inhibit Cu2+/Zn2+-induced neurotoxicity. Initially, we examined the protective effect of various organic acids on Cu2+/Zn2+-induced neurotoxicity, and found that pyruvic acid clearly suppresses Cu2+/Zn2+-induced neurotoxicity in GT1-7 cells. Next, we examined the protective mechanisms of pyruvic acid against Cu2+/Zn2+-induced neurotoxicity. Specifically, we examined the possibilities that pyruvic acid chelates Cu2+ and Zn2+ or suppresses the ER stress response, but found that neither was suppressed by pyruvic acid treatment. In contrast, pyruvic acid significantly suppressed cytochrome c release into cytoplasm, an index of mitochondrial injury, in a dose-dependent manner. These results suggest that pyruvic acid prevents Cu2+/Zn2+-induced neuronal cell death by suppressing mitochondrial injury. Based on our results, we assume that pyruvic acid may be therapeutically beneficial for neurological diseases involving neuronal cell death such as vascular dementia.

Characterization of the dynamic change of microRNA expression in mice hypothalamus during the time of female puberty

Puberty onset is a milestone in sexual development. A tumor suppress gene (TSG) network had been reported to be involved in the regulation of female puberty onset. The observations in rodents and primates showed a potential link between microRNAs and puberty onset. To figure out what miRNAs play roles in this important biological process, profilings of microRNAs in the hypothalamus of female mice from three different pubertal stages, juvenile [postnatal day (P10)], early pubertal (P25) and pubertal (P30) were performed on the Affymetrix GeneChip miRNA 3.0 Arrays, the cerebral cortex (CTX) was used as a control tissue. 20 miRNAs were shown to be differentially expressed in hypothalamus (fold change > 1.5, P < 0.05), but not in CTX during the transition from juvenile to pubertal. Four of them were validated by real-time quantitative RT-PCR (qRT-PCR) method. 1018 genes were predicted as the targets of these miRNAs. Further bioinformatics analysis suggested that these target genes were involved in many important signaling pathways, especially in the cancer related pathways. We also found that about 90% of these target genes were expressed in the hypothalamus, as well as in the immortalized GnRH-producing GT1-7 cells, which provided additional evidence that these miRNAs could be female puberty onset related. Here we present a novel comprehensive data set of miRNA gene expression during the puberty onset; and it provides an important recourse for the future functional characterization of individual miRNAs and their targets in mouse hypothalamus and in GT1-7 cells.

Effects of N-carbamylglutamate and L-arginine on gonadotrophin-releasing hormone (GnRH) gene expression and secretion in GT1-7 cells.

Recent studies have shown that N-carbamylglutamate (NCG) and arginine (ARG) supplementation improves reproductive performance in livestock. The objectives of the present study were to evaluate the effects of NCG and ARG on GT1-7 cell gonadotrophin-releasing hormone (GnRH) secretion, gene expression and cell proliferation. GT1-7 cells were treated in vitro with different concentrations of NCG (0-1.0mM) or ARG (0-4.0mM) in serum-free medium for 12 or 24h. For GnRH secretion and cell proliferation, GT1-7 cells were more sensitive to NCG than ARG. NCG treatment after 12h increased cell numbers and inhibited GnRH secretion in a dose-dependent manner (P<0.05), although there was no significant effect of NCG on these parameters after 24h culture. ARG treatment decreased GnRH secretion after 24h (P<0.05), whereas it had no effect after 12h. GT1-7 cells express GnRH, Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), neuronal nitric oxide synthase (nNOS) and estrogen receptor α (ERα) genes. High concentrations of NCG (1.0mM) and ARG (4.0mM) inhibited (P<0.05) GnRH and nNOS mRNA abundance in GT1-7 cells. ARG treatment decreased Kiss1 and increased ERα mRNA abundance. Thus, high concentrations of NCG (1.0mM) and ARG (4.0mM) may act both directly and indirectly to regulate GnRH neuron function by downregulating genes related to GnRH synthesis and secretion to slow GnRH production while stimulating GT1-7 cell proliferation.

Inhibition of central de novo ceramide synthesis restores insulin signaling in hypothalamus and enhances β-cell function of obese Zucker rats

ObjectivesHypothalamic lipotoxicity has been shown to induce central insulin resistance and dysregulation of glucose homeostasis; nevertheless, elucidation of the regulatory mechanisms remains incomplete. Here, we aimed to determine the role of de novo ceramide synthesis in hypothalamus on the onset of central insulin resistance and the dysregulation of glucose homeostasis induced by obesity. MethodsHypothalamic GT1-7 neuronal cells were treated with palmitate. De novo ceramide synthesis was inhibited either by pharmacological (myriocin) or molecular (si-Serine Palmitoyl Transferase 2, siSPT2) approaches. Obese Zucker rats (OZR) were intracerebroventricularly infused with myriocin to inhibit de novo ceramide synthesis. Insulin resistance was determined by quantification of Akt phosphorylation. Ceramide levels were quantified either by a radioactive kinase assay or by mass spectrometry analysis. Glucose homeostasis were evaluated in myriocin-treated OZR. Basal and glucose-stimulated parasympathetic tonus was recorded in OZR. Insulin secretion from islets and β-cell mass was also determined. ResultsWe show that palmitate impaired insulin signaling and increased ceramide levels in hypothalamic neuronal GT1-7 cells. In addition, the use of deuterated palmitic acid demonstrated that palmitate activated several enzymes of the de novo ceramide synthesis pathway in hypothalamic cells. Importantly, myriocin and siSPT2 treatment restored insulin signaling in palmitate-treated GT1-7 cells. Protein kinase C (PKC) inhibitor or a dominant-negative PKCζ also counteracted palmitate-induced insulin resistance. Interestingly, attenuating the increase in levels of hypothalamic ceramides with intracerebroventricular infusion of myriocin in OZR improved their hypothalamic insulin-sensitivity. Importantly, central myriocin treatment partially restored glucose tolerance in OZR. This latter effect is related to the restoration of glucose-stimulated insulin secretion and an increase in β-cell mass of OZR. Electrophysiological recordings also showed an improvement of glucose-stimulated parasympathetic nerve activity in OZR centrally treated with myriocin. ConclusionOur results highlight a key role of hypothalamic de novo ceramide synthesis in central insulin resistance installation and glucose homeostasis dysregulation associated with obesity.

Thioredoxin-albumin fusion protein prevents copper enhanced zinc-induced neurotoxicity via its antioxidative activity

Zinc (Zn) is a co-factor for a vast number of enzymes, and functions as a regulator for immune mechanism and protein synthesis. However, excessive Zn release induced in pathological situations such as stroke or transient global ischemia is toxic. Previously, we demonstrated that the interaction of Zn and copper (Cu) is involved in the pathogenesis of Alzheimer’s disease and vascular dementia. Furthermore, oxidative stress has been shown to play a significant role in the pathogenesis of various metal ions induced neuronal death. Thioredoxin-Albumin fusion (HSA-Trx) is a derivative of thioredoxin (Trx), an antioxidative protein, with improved plasma retention and stability of Trx. In this study, we examined the effect of HSA-Trx on Cu2+/Zn2+-induced neurotoxicity. Firstly, HSA-Trx was found to clearly suppress Cu2+/Zn2+-induced neuronal cell death in mouse hypothalamic neuronal cells (GT1-7 cells). Moreover, HSA-Trx markedly suppressed Cu2+/Zn2+-induced ROS production and the expression of oxidative stress related genes, such as heme oxygenase-1. In contrast, HSA-Trx did not affect the intracellular levels of both Cu2+ and Zn2+ after Cu2+/Zn2+ treatment. Finally, HSA-Trx was found to significantly suppress endoplasmic reticulum (ER) stress response induced by Cu2+/Zn2+ treatment in a dose dependent manner. These results suggest that HSA-Trx counteracted Cu2+/Zn2+-induced neurotoxicity by suppressing the production of ROS via interfering the related gene expressions, in addition to the highly possible radical scavenging activity of the fusion protein. Based on these findings, HSA-Trx has great potential as a promising therapeutic agent for the treatment of refractory neurological diseases.

Transcriptional interaction between cFOS and the homeodomain-binding transcription factor VAX1 on the GnRH promoter controls Gnrh1 expression levels in a GnRH neuron maturation specific manner

Gonadotropin-releasing hormone (GnRH) is required for pubertal onset and reproduction, thus the control of GnRH transcription is tightly regulated during development and adulthood. GnRH neuron development depends on transcription factors of the homeodomain family. For example, Ventral anterior homeobox 1 (Vax1) is necessary to maintain GnRH expression after embryonic day 13 in the mouse. To further our understanding of the mechanisms by which VAX1 regulates GnRH gene expression, we asked whether VAX1 interacts with other transcription factors to modify GnRH expression levels. Using the GnRH cell lines, GN11 and GT1-7, we found that activation of PKC enhances expression of the immediate early gene cFos in both GN11, and GT1-7, and represses expression of Vax1 in GT1-7. Further, VAX1 interacts with cFOS while bound to the GnRH promoter. In immature GN11 cells, VAX1 and cFOS enhance GnRH expression, whereas VAX1 and cFOS have a repressive role in the mature GT1-7 cells.

The alpha-7 nicotinic acetylcholine receptor is involved in a direct inhibitory effect of nicotine on GnRH release: In vitro studies

The activation of nicotinic cholinergic receptors (nAChR) inhibits the reproductive axis; however, it is not clear whether nicotine may directly modulate the release of hypothalamic gonadotropin-releasing hormone (GnRH). Experiments carried out in GT1-1 immortalized GnRH neurons reveal the presence of a single class of high affinity α4β2 and α7 nAchR subtypes. The exposure of GT1-1 cells to nicotine does not modify the basal accumulation of GnRH. However, nicotine was found to modify GnRH pulsatility in perifusion experiments and inhibits, the release of GnRH induced by prostaglandin E1 or by K+-induced cell depolarization; these effects were reversed by D-tubocurarine and α-bungarotoxin. In conclusion, the results reported here indicate that: functional nAChRs are present on GT1-1 cells, the activation of the α-bungarotoxin-sensitive subclass (α7) produces an inhibitory effect on the release of GnRH and that the direct action of nicotine on GnRH neurons may be involved in reducing fertility of smokers.