High fat induces acute and chronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-α on appetite-regulating NPY neurons.

BACKGROUND: Consumption of dietary fat is one of the key factors leading to obesity. High-fat diet (HFD)-induced obesity is characterized by induction of inflammation in the hypothalamus; however, the temporal regulation of proinflammatory markers and their impact on hypothalamic appetite-regulating neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons remains undefined. METHODS: Mice were injected with an acute lipid infusion for 24 h or fed a HFD over 8-20 weeks. Characterized mouse NPY/AgRP hypothalamic cell lines were used for in vitro experimentation. Immunohistochemistry in brain slices or quantitative real-time PCR in cell lines, was performed to determine changes in the expression of key inflammatory markers and neuropeptides. RESULTS: Hypothalamic inflammation, indicated by tumor necrosis factor (TNF)-α expression and astrocytosis in the arcuate nucleus, was evident following acute lipid infusion. HFD for 8 weeks suppressed TNF-α, while significantly increasing heat-shock protein 70 and ciliary neurotrophic factor, both neuroprotective components. HFD for 20 weeks induced TNF-α expression in NPY/AgRP neurons, suggesting a detrimental temporal regulatory mechanism. Using NPY/AgRP hypothalamic cell lines, we found that palmitate provoked a mixed inflammatory response on a panel of inflammatory and endoplasmic reticulum (ER) stress genes, whereas TNF-α significantly upregulated IκBα, nuclear factor (NF)-κB and interleukin-6 mRNA levels. Palmitate and TNF-α exposure predominantly induced NPY mRNA levels. Utilizing an I kappa B kinase ß (IKKß) inhibitor, we demonstrated that these effects potentially occur via the inflammatory IKKß/NF-κB pathway. CONCLUSIONS: These findings indicate that acute lipid and chronic HFD feeding in vivo, as well as acute palmitate and TNF-α exposure in vitro, induce markers of inflammation or ER stress in the hypothalamic appetite-stimulating NPY/AgRP neurons over time, which may contribute to a dramatic alteration in NPY/AgRP content or expression. Acute and chronic HFD feeding in vivo temporally regulates arcuate TNF-α expression with reactive astrocytosis, which suggests a time-dependent neurotrophic or neurotoxic role of lipids.

Estrogen inhibits NPY secretion through membrane-associated estrogen receptor (ER)-α in clonal, immortalized hypothalamic neurons.

OBJECTIVE: Estrogen (E(2)) has an inhibitory effect on food intake by acting centrally in the hypothalamus, although it is not clear which hypothalamic neurons are involved in this process. Earlier studies from our lab and others have implicated neuropeptide Y (NPY) as an important central anorexigenic target of E(2). This study was designed to investigate whether E(2) can directly regulate NPY secretion and examine the cellular mechanisms and receptors responsible for this anorexigenic action of E(2). DESIGN: Clonal, murine, hypothalamic neuronal cell models, mHypoE-42 and mHypoA-2/12, were investigated for NPY secretory responses to 17ß-estradiol (E(2)) in the presence or absence of pharmacological inhibitors directed against the phosphatidylinositol-3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and AMP-activated kinase (AMPK) pathways or to estrogen receptor (ER) specific agonists/antagonists. MEASUREMENTS: The presence of hypothalamic markers and characterization of neuronal cell lines was completed with polymerase chain reaction. NPY levels were measured using an enzyme immunoassay (EIA). The expression of ER-α and caveolin-1 was analyzed using immunocytochemistry. RESULTS: E(2) significantly decreased NPY secretion in both the mHypoE-42 and mHypoA-2/12 neurons. The E(2)-mediated repression of NPY secretion in the mHypoE-42 and mHypoA-2/12 neurons required ER-α, but not ER-ß, as shown by studies using an ER-specific agonist/antagonists. Additionally, using immunocytochemistry we detected colocalization of ER-α and the membrane-associated signaling protein caveolin-1. Importantly, using E(2)-conjugated bovine serum albumin (E(2)-BSA) and ER antagonists, we were able to show that the E(2)-mediated decrease in NPY secretion occurred through membrane-bound ER-α. Finally, using a combination of pharmacological inhibitors, we found that inhibition of the PI3K or AMPK pathway blocked the E(2)-mediated decrease in NPY secretion. CONCLUSION: These findings indicate that the central anorexigenic action of E(2) occurs at least partially through hypothalamic NPY-synthesizing neurons. This regulation of NPY secretion occurs through rapid signaling mechanisms through membrane bound ER-α.

The generation of an array of clonal, immortalized cell models from the rat hypothalamus: analysis of melatonin effects on kisspeptin and gonadotropin-inhibitory hormone neurons.

Significant information on reproductive function has been generated based on the rat model, including many seminal discoveries. Yet little is known about the molecular and cellular events involved in control of reproductive function, mainly due to the pervasive lack of cell models from rat. We have therefore generated a wide array of cell lines using primary cell culture from the rat hypothalamus. Immortalization of the primary cells was achieved through retroviral transfer of T-antigen, followed by selection with geneticin. The mixed cell populations were subcloned and each clonal cell line was analyzed for expression of specific cellular markers. Each line has a distinct phenotypic profile, with expression of key neuroendocrine markers. We have functionally analyzed two clonal cell lines, rHypoE-7 and rHypoE-8, for hormones implicated in the control of gonadotropin-releasing hormone neuronal function through melatonin, specifically kisspeptin (KISS) and RF-amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibiting hormone, GnIH). We detected functional melatonin receptor activity, as each cell line exhibited inhibition of forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation. Upon treatment with 10 nM melatonin, we found that KISS gene expression was decreased in the rHypoE-8 cell line, while RFRP-3 was increased in the rHypoE-7 cell line. These results are in accordance with the differential regulatory functions of these two peptides, particularly on GnRH neuronal control. These cell lines will serve as novel tools for the analysis of the cellular and molecular mechanisms involved in hypothalamic control of a number of physiological processes described in the rat animal model.

Hypothalamic preproghrelin gene expression is repressed by insulin via both PI3-K/Akt and ERK1/2 MAPK pathways in immortalized, hypothalamic neurons.

The gut peptide ghrelin is expressed within neurons of the hypothalamus. Using a hypothalamic cell line, mHypoE-38 neurons, the effect of insulin on preproghrelin gene expression was assayed. These cells contain neuron-specific markers, preproghrelin and the insulin receptor. We determined that insulin has direct effects on preproghrelin gene expression. Insulin (10 nM) stimulated protein kinase B (Akt) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation from 5 to 60 min and 5 min, respectively, and led to repression of preproghrelin gene expression at 2 h. Pharmacological inhibitors to phosphoinositide-3-kinase (PI3-K; LY294002) and MEK (PD98059) demonstrated that basal ghrelin gene expression is regulated by the PI3-K pathway and requires the mitogen-activated protein kinase pathway for insulin-stimulated preproghrelin repression. These results demonstrate that insulin has a direct effect on hypothalamic neurons to decrease preproghrelin gene expression through classic insulin pathways.

Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression.

The physiological actions of nitric oxide (NO) as a signaling molecule in endothelial and brain cells and as a toxic molecule used by activated immune cells have been the focus of a wide range of studies. Nevertheless, the downstream effector molecules of this important neuromodulator are not well understood. We have previously demonstrated that expression of the gene for the reproductive neuropeptide, GnRH, is repressed by the glutamate/NO/cyclic GMP (cGMP) signal transduction pathway through cGMP-dependent protein kinase in the hypothalamic GnRH-secreting neuronal cell line GT1-7. This repression localized within a previously characterized 300-bp neuron-specific enhancer. Here, we find that mutation of either of two adjacent elements within the enhancer eliminates repression by this pathway. An AT-rich sequence located at -1695 has homology to the octamer motif known to bind POU-homeodomain proteins, while the adjacent element at -1676 has homology to the C/EBP (CCAAT/enhancer-binding protein) protein family consensus sequence. Antibody supershift assays reveal that one of the proteins bound at the -1695 sequence is Oct-1, and one of the proteins bound to the element at -1676 is C/EBPbeta. These two proteins can bind simultaneously to the adjacent -1695 and -1676 binding sites in vitro. In nuclear extracts of GT1-7 cells treated with an NO donor, the intensity of the Oct-1 complex is increased. However, although Western blot analysis indicates that neither Oct-1 nor C/EBPbeta protein levels are increased, the relative binding affinity of Oct-1 is increased. Dephosphorylation of the nuclear extracts decreases binding of the Oct-1 complex to the -1695 site only in NO donor-treated extracts. Thus, we conclude that Oct-1 and C/EBPbeta are both downstream transcriptional regulators involved in the repression of GnRH gene expression by the glutamate/NO/ cGMP signal transduction pathway.

Estrogen directly respresses gonadotropin-releasing hormone (GnRH) gene expression in estrogen receptor-alpha (ERalpha)- and ERbeta-expressing GT1-7 GnRH neurons.

Estrogen has wide-ranging and complex effects on the reproductive axis, which are often difficult to interpret from in vivo studies. Estrogen negatively regulates tonic GnRH synthesis and also plays a pivotal role in the positive regulation of GnRH necessary for the preovulatory surge. To dissect the mechanisms by which these divergent effects occur, we attempted to observe the direct action of estrogen on the regulation of GnRH messenger RNA (mRNA) levels using the well characterized, GnRH-secreting, hypothalamic cell line, GT1-7. Using RT-PCR, we first investigated estrogen receptor transcript expression in GT1-7 neurons. We found that the GT1-7 cells express both estrogen receptor-alpha (ERalpha) and the recently described ERbeta mRNAs. We also detected the presence of both receptor subtypes in the GT1-7 neurons by Western blot analysis using specific ER antibodies. By Northern blot analysis of total GT1-7 RNA, we found that 17beta-estradiol (1 nM) down-regulates GnRH mRNA levels to approximately 55% of basal levels over a 48-h time course. This effect appears to occur specifically through an ER-mediated mechanism, as ICI 182,780, a complete ER antagonist, blocks the repression of GnRH mRNA levels by estradiol. The recently reported ERalpha-specific agonist/ERbeta-specific antagonist 2,2-bis-(p-hydroxyphenyl-1,1,1-trichloroethane (HPTE), a methoxychlor metabolite, also down-regulated GnRH gene expression. The repression of GnRH mRNA levels appears to occur at the transcriptional level, as simian virus 40 T antigen mRNA expression, which is under the control of 2.3 kb of the rat GnRH 5'-regulatory region, mimics the down-regulation of GnRH after treatment with estradiol. As the rat GnRH regulatory region in GT1-7 neurons does not appear to harbor a classic estrogen response element, the mechanism involved in the repression of GnRH has yet to be determined. These results suggest that estradiol directly regulates GnRH gene expression at the level of the GnRH neuron and may exert its neuroendocrine control through direct interaction with specific receptors expressed in these cells.

Regulation of gonadotropin-releasing hormone (GnRH) gene expression by 5alpha-dihydrotestosterone in GnRH-secreting GT1-7 hypothalamic neurons.

Hypothalamic GnRH secretory neurons are precisely regulated by circulating gonadal steroids. However, the question of whether these cells are directly responsive to steroid hormones remains a central and controversial issue in reproductive science. In the present study, we demonstrate the expression of androgen receptor (AR) in a mouse hypothalamic GnRH-secreting cell line, GT1-7. AR messenger RNA was detected by Northern blot analysis of 10 microg total cellular RNA. Western blot analysis revealed a 110K AR immunoreactive band, and saturation binding analysis confirmed the presence of a high affinity low capacity androgen binding entity (Kd = 0.06 nM; Bmax = 12.4 fmol/mg protein). In addition, GT1-7 cells were found to express ARA70, an AR-specific coactivator that has been reported to enhance transactivational activity of the AR. GT1-7 cells transiently transfected with an androgen responsive MMTV-luciferase reporter construct displayed a 4.2-fold induction of luciferase reporter gene activity by 1 nM 5alpha-dihydrotestosterone (DHT), further demonstrating the presence of a functional AR. Treatment of GT1-7 cells with 1 or 10 nM DHT resulted in approximately 55% reduction in GnRH messenger RNA measured at 24 and 36 h after treatment. This repression was completely blocked by hydroxyflutamide, an AR antagonist. These results provide the first demonstration that androgen acts directly through an AR-mediated pathway to repress GnRH gene expression in hypothalamic GnRH-secreting neurons.

NMDA and nitric oxide act through the cGMP signal transduction pathway to repress hypothalamic gonadotropin-releasing hormone gene expression.

The key roles of the excitatory neurotransmitter glutamate and its second messengers, nitric oxide (NO) and cGMP, in long-term potentiation and neural plasticity are well documented. However, complex functions such as memory are likely to require long term changes in synaptic efficacy which require gene expression and protein synthesis. Here we demonstrate that the glutamate receptor agonist, N-methyl-D-aspartic acid (NMDA), nitric oxide (NO) and cGMP each repress expression of the gonadotropin-releasing hormone (GnRH) gene in the hypothalamic cell line, GT1. This repression is dependent upon signals from NMDA receptors activating NO synthase to synthesize NO. In turn NO induces guanylyl cyclase to synthesize cGMP, activating cGMP- dependent protein kinase. Repression requires elevation of calcium because it only occurs in the presence of calcium ionophore or with release of intracellular calcium. Repression also requires protein synthesis. Activation of this pathway specifically represses expression of a reporter gene containing the regulatory region of the GnRH gene in transfected GT1 cells, indicating that repression occurs at the transcriptional level. Furthermore the target for transcriptional repression is a 300 bp neuron-specific enhancer found 1.5 kb upstream of the GnRH gene which is sufficient to confer repression to a heterologous promoter. Thus the NMDA/NO/cGMP neurotransmitter signal transduction pathway controls not only synaptic function but also neuron-specific gene expression.

A neuron-specific enhancer targets expression of the gonadotropin-releasing hormone gene to hypothalamic neurosecretory neurons.

The molecular mechanisms specifying gene expression in individual neurons of the mammalian central nervous system have been difficult to study due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons. We have developed clonal, differentiated, neuronal tumor cell lines of the hypothalamic GnRH-producing neurons by targeting tumorigenesis in transgenic mice. These cells (GT1 cells) provide a model system for molecular studies of GnRH gene regulation. Here we present the identification and characterization of a neuron-specific enhancer responsible for directing expression of the rat GnRH gene in GT1 hypothalamic neurons. This approximately 300 base pair (bp) upstream region (-1571 to -1863) confers enhancer activity to a short -173-bp GnRH promoter or to a heterologous promoter only in GT1 cells. The enhancer is bound by multiple GT1 nuclear proteins over its entire length. Deletion of more than 30 bp from either end dramatically reduces activity, and even large internal fragments carrying seven of the eight DNAse I-protected elements show decreased activity. Scanning replacement mutations demonstrate that several of the internal elements are required for activity of the enhancer. Thus, the GnRH gene is targeted to hypothalamic neurons by a complex multicomponent enhancer that relies on the interaction of multiple nuclear-protein binding enhancer elements.