Mouse Hypothalamic Neuronal Cell Research Articles

Hypothalamic miR-1983 Targets Insulin Receptor β and the Insulin-mediated miR-1983 Increase Is Blocked by Metformin.

MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor β subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease.

Estrogen Regulates the Trafficking of Leptin Receptors

During menopause, when endogenous estrogen levels are depleted, a rise in hypertension is observed in 75% of post-menopausal women, suggesting there is a cardioprotective effect from estrogen. In previous studies, estrogen has been shown to effect hypothalamic paraventricular nuclei by its action to lessen the impact of a glutamate induced increase in blood pressure. These data, along with others, suggest that the cardioprotective effect of estrogen is regionally specific in the central nervous system. However, the mechanism(s) behind estrogen's attenuating effect on blood pressure have not been fully elucidated. We propose that estrogen regulates blood pressure in part, by inhibiting the actions of leptin. Leptin is an adipocyte-derived peptide hormone. Activation of its signaling pathway within the central nervous system can alter blood pressure. Studies with injections of leptin in rodent's dorsomedial hypothalamus showed an increase in blood pressure, while antagonism of leptin receptors correlated with diminished blood pressure. Both results suggest that adjustment of leptin signaling in the dorsomedial hypothalamus is sufficient to alter blood pressure. In this study, we hypothesize that estrogen can block leptin receptor activation and downstream signaling events by increasing the expression of SOCS3 (suppressor of cytokine signaling 3), a negative modulator of leptin signaling. We tested our hypothesis by utilizing an in vitro system (i.e. GT1-7 mouse hypothalamic neuronal cells) and an in vivo system (i.e. female C57B6 mice) that were treated with estradiol (100nM). Our data demonstrated that administering estrogen decreased the expression of leptin receptors in the hypothalamus and we proposed this occurred due to the increase in SOCS3 expression we observed when estradiol was administered. Taken together, these data suggest that estrogen can attenuate leptin's physiological response on blood pressure by increasing an inhibitor (i.e. SOCS3) of leptin's signaling.

Apelin Receptor Signaling Protects GT1-7 GnRH Neurons Against Oxidative Stress In Vitro.

Hypothalamic-pituitary-adrenal (HPA) axis regulates stress response in the body and abnormal increase in oxidative stress contributes to the various disease pathogenesis. Although hypothalamic distribution of Apelin receptor (APLNR) has been studied, the potential regulatory role in hormone releasing function of hypothalamus in response to stress is not well elucidated yet. To determine whether APLNR is involved in the protection of the hypothalamus against oxidative stress, gonadotropin-releasing hormone (GnRH) cells were used as an in vitro model system. GT1-7 mouse hypothalamic neuronal cell line was subjected to H2O2 and hypoxia induced oxidative stress under various circumstances including APLNR overexpression, knockdown and knockout. Overexpression and activation of APLNR in GnRH producing neurons caused an increase in cell proliferation under oxidative stress. In addition, blockage of APLNR function by siRNA reduced GnRH release. Activation of APLNR initiated AKT kinase pathway as a proliferative response against hypoxic culture conditions and blocked apoptosis. Although expression and activation of APLNR have not been related to GnRH neuron differentiation during development, positive contribution of activated APLNR signaling to GnRH release in mouse embryonic stem cell derived GnRH neurons was observed in the present study. Sustained overexpression and complete deletion of APLNR in mouse embryonic stem cell derived GnRH neurons reduced GnRH release in vitro. The present findings suggest that expression and activation of APLNR in GnRH releasing GT1-7 neurons might induce a protective mechanism against oxidative stress induced cell death and APLNR signaling may play a role in GnRH neurons.

Direct effects of antipsychotic drugs on insulin, energy sensing and inflammatory pathways in hypothalamic mouse neurons

IntroductionSecond-generation antipsychotics cause serious metabolic side effects, but the mechanisms behind these effects remain largely unknown. However, emerging evidence supports that antipsychotics may act upon the hypothalamus, the primary brain region understood to regulate energy homeostasis. We have recently reported that the antipsychotics olanzapine, clozapine, and aripiprazole can directly act on hypothalamic rat neurons (rHypoE-19) to impair insulin, energy sensing, and modulate inflammatory pathways. In the current paper, we sought to replicate these findings to a mouse neuronal model. MethodsThe mouse hypothalamic neuronal cell line, mHypoE-46, was treated with olanzapine, clozapine, or aripiprazole. Western blots were used to measure the energy sensing protein AMPK, components of the insulin signalling pathway (AKT, GSK3β), and components of the MAPK pathway (ERK1/2, JNK, p38), the latter linked to inflammation. RT-qPCR was used to measure mRNA expression of the inflammatory mediators IL-6, IL-10, and BDNF, well as putative receptors in the mHypoE-46 (current) and the rHypoE-19 (previously studied) cell lines. ResultsIn the mHypoE-46 neurons, olanzapine and aripiprazole increased AMPK phosphorylation, while clozapine and aripiprazole inhibited insulin-induced phosphorylation of AKT. Clozapine increased JNK and aripiprazole decreased ERK1/2 phosphorylation. Olanzapine also decreased IL-6 mRNA expression, while olanzapine and clozapine increased IL-10 mRNA expression. The rHypoE-19 neurons expressed the H1, 5 H T2A, and M3 receptors, while the mHypoE-46 neurons expressed the 5 H T2A, D2, and M3 receptors. Neither cell line expressed the 5 H T2C receptor. ConclusionSimilar to observed effects of these agents in rat neurons, induction of AMPK by aripiprazole and olanzapine suggests impaired energy sensing, while suppression of insulin-induced pAKT by clozapine and aripiprazole suggests impaired insulin signalling, seen across both rodent derived hypothalamic cell lines. Conversely, olanzapine-induced suppression of pro-inflammatory IL-6, alongside olanzapine and clozapine-induced IL-10, demonstrate anti-inflammatory effects, which do not corroborate with our prior observations in the rat neuronal line. The different findings between cell lines could be explained by differential expression of neurotransmitters receptors and/or reflect genetic heterogeneity across the rat and mouse lines. However, overall, our findings support direct effects of antipsychotics to impact insulin, energy sensing, and inflammatory pathways in hypothalamic rodent neurons.

Deficient ganglioside synthesis restores responsiveness to leptin and melanocortin signaling in obese KKAy mice

GM3, a precursor for synthesis of a- and b-series gangliosides, is elevated in adipocytes of obese model animals and in sera of obese human patients with type 2 diabetes and/or dyslipidemia. GM3 synthase (GM3S)-KO C57BL/6 mice display enhanced insulin sensitivity and reduced development of high-fat diet-induced insulin resistance. However, the pathophysiological roles of GM3 and related gangliosides in the central control of feeding and metabolism remain unclear. We found that a mouse model (KKAy GM3S KO) generated by KO of the GM3S gene in the yellow obese strain, KKAy, displayed significant amelioration of obese phenotype. Whereas KKAy mice were hyperphagic and developed severe obesity, KKAy GM3S KO mice had significantly lower body weight and food intake, and greater glucose and insulin tolerance. The hypothalamic response to intraperitoneal administration of leptin was greatly reduced in KKAy mice, but was retained in KKAy GM3S KO mice. In studies of a cultured mouse hypothalamic neuronal cell line, enhanced leptin-dependent phosphorylation of ERK was observed in GM3S-deficient cells. Furthermore, KKAy GM3S KO mice did show altered coat color, suggesting that GM3S is also involved in melanocortin signaling. Our findings, taken together, indicate that GM3-related gangliosides play key roles in leptin and melanocortin signaling.

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.

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.

The effects of L-theanine and EGCG on palmitic acid induced inflammation in mouse hypothalamic neuronal cell lines (mHypoE-N42)

The effects of L-theanine and EGCG on palmitic acid induced inflammation in mouse hypothalamic neuronal cell lines (mHypoE-N42)

Xenin-induced feeding suppression is not mediated through the activation of central extracellular signal-regulated kinase signaling in mice

Xenin is a gut hormone that reduces food intake by partly acting through the hypothalamus via neurotensin receptor 1 (Ntsr1). However, specific signaling pathways that mediate xenin-induced feeding suppression are not fully understood. Activation of Ntsr1 leads to the activation of the extracellular signal-regulated kinase (ERK). Hypothalamic ERK participates in the regulation of food intake by mediating the effect of hormonal signals. Therefore, we hypothesized that the anorectic effect of xenin is mediated by hypothalamic ERK signaling. To address this hypothesis, we compared levels of phosphorylation of ERK1/2 (pERK1/2) in the hypothalamus of both control and xenin-treated mice. The effect of xenin on ERK1/2 phosphorylation was also examined in mouse hypothalamic neuronal cell lines with or without Ntsr1. We also examined the effect of the blockade of central ERK signaling on xenin-induced feeding suppression in mice. The intraperitoneal (i.p.) injection of xenin caused a significant increase in the number of pERK1/2-immunoreactive cells in the hypothalamic arcuate nucleus. The majority of pERK1/2-positive cells expressed neuronal nuclei (NeuN), a marker for neurons. Xenin treatment increased pERK1/2 levels in one cell line expressing Ntsr1 but not another line without Ntsr1 expression. Both i.p. and intracerebroventricular (i.c.v.) injections of xenin reduced food intake in mice. The i.c.v. pre-treatment with U0126, a selective inhibitor of ERK1/2 upstream kinases, did not affect xenin-induced reduction in food intake. These findings suggest that although xenin activates ERK signaling in subpopulations of hypothalamic neurons, xenin does not require the activation of hypothalamic ERK signaling pathway to elicit feeding suppression.

Aromatase promoter I.f is regulated by progesterone receptor in mouse hypothalamic neuronal cell lines

Aromatase catalyzes the conversion of C(19) steroids to estrogens. Aromatase and progesterone, both of which function at different steps of steroidogenesis, are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The aromatase gene (Cyp19a1) is selectively expressed in distinct neurons of the mouse hypothalamus through a distal brain-specific promoter, I.f, located ∼40 kb upstream of the coding region. However, the regulation of aromatase expression in the brain is not well understood. In this study, we investigated a short feedback effect of progesterone analogues on aromatase mRNA expression and enzyme activity in estrogen receptor α (Esr1)-positive or -negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. In a hypothalamic neuronal cell line that highly expresses aromatase, progesterone receptor (Pgr), and Esr1, a progesterone agonist, R5020, inhibited aromatase mRNA level and enzyme activity. The inhibitory effect of R5020 was reversed by its antagonist, RU486. Deletion mutants of promoter I.f suggested that inhibition of aromatase expression by progesterone is conferred by the nt -1000/-500 region, and R5020 enhanced binding of Pgr to the nt -800/-600 region of promoter I.f. Small interfering RNA knockdown of Pgr eliminated progesterone-dependent inhibition of aromatase mRNA and enzyme activity. Taken together, progesterone enhances recruitment of Pgr to specific regions of the promoter I.f of Cyp19a1 and regulates aromatase expression in hypothalamic neurons.

Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines1

Aromatase (CYP19A1) catalyzes the conversion of C(19) steroids to estrogens. Aromatase and its product estradiol (E(2)) are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The regulation of aromatase expression in the brain is not well understood. The aromatase (Cyp19a1) gene is selectively expressed in distinct neurons of the hypothalamus through a distal brain-specific promoter I.f located approximately 36 kb upstream of the coding region. Here, we investigated a short feedback effect of E(2) on aromatase mRNA expression and enzyme activity using estrogen receptor alpha (ESR1; also known as ER alpha)-positive or ESR1-negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. Estradiol regulated aromatase mRNA expression and enzyme activity in a time- and dose-dependent manner, whereas an E(2) antagonist reversed these effects. The nucleotide -200/-1 region of promoter I.f conferred E(2) responsiveness. Two activator protein 1 (AP-1) elements in this region were essential for induction of promoter activity by E(2). ESR1 and JUN (c-Jun) bound to these AP-1 motifs in intact cells and under cell-free conditions. The addition of an ESR1 mutant that interacts with JUN but not directly with DNA enhanced E(2)-dependent promoter I.f activity. Independently, we demonstrated an interaction between ESR1 and JUN in hypothalamic cells. Knockdown of ESR1 abolished E(2)-induced aromatase mRNA and enzyme activity. Taken together, E(2) regulates Cyp19a1 expression via promoter I.f by enhanced binding of an ESR1/JUN complex to distinct AP-1 motifs in hypothalamic cells. We speculate that this mechanism may, in part, regulate gonadotropin secretion and sexual activity.

Fine Tuning PDK1 Activity by Phosphorylation at Ser163

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.

Protective effects of Orostachys japonicus A. Berger (Crassulaceae) on H 2O 2-induced apoptosis in GT1-1 mouse hypothalamic neuronal cell line

Oxidative stress is a major cause of neurodegenerative diseases, so the protection of neuronal cells from reactive oxygen species can be beneficial for the prevention and treatment of these diseases. Methanol extract of Orostachys japonicus A. Berger (Crassulaceae), a traditional oriental medical herb, was shown to have a protective effect on H 2O 2-induced apoptosis in GT1-1 mouse hypothalamic neuronal cell line which was detected by flow cytometry after propidium iodide staining. Among fractions of O. japonicus, chloroform fraction had the highest protective effect, and water fraction had no protective effect suggesting that the active ingredients might be hydrophobic compounds.