Unknown,Transcriptomics,Genomics,Proteomics

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Regulation of pituitary gene expression by adrenalectomy

ABSTRACT: Overproduction of adrenal hormones causes obesity and hypertension, whereas adrenalectomy (ADX) leads to weight loss and hypotension. The pituitary gland is a major target organ for the adrenal hormones. To identify potential mediators for the effect of ADX on pituitary system regulating weight loss and hypotension, we investigated the effect of ADX on pituitary gene expression using serial analysis of gene expression (SAGE). The numbers of analyzed SAGE tag were 126688 tags for intact mice (n = 51) and 59482 tags for ADX mice (n = 12), which corresponded to 45151 and 21790 distinct tag species, respectively. Thirty-three pituitary genes were differentially expressed between intact and ADX mice. Three genes encoding for pro-opiomelanocortin have been upregulated by ADX. The ADX has induced the gene expression of growth hormone while downregulating an expressed sequence tag similar to (EST) growth hormone. The ADX reduced the expression of genes involved in mitochondrial oxidative phosphorylation, such as NADH dehydrogenase 3 and cytochrome c oxidase 3, whereas neuromedin B gene were dramatically induced. In addition, the ADX increased the expression levels of other three genes involved in hormone release (response to metastatic cancers 1), extracellular matrix (matrix gamma-carboxyglutamate gla protein), and cation transport (solute carrier family member 17). Moreover, one functionally-uncharacterized and 20 novel transcripts were significantly modulated by ADX. Thus the current study has revealed the alterations in the pituitary gene expression that may play key roles in the mechanisms of ADX-induced weight loss and hypotension. Keywords: adrenalectomy, serial analysis of gene expression Rodents and pituitary gland sampling. Male C57BL6 mice were obtained from Charles River Laboratories (St. Constant, QC, Canada), at 12-14 weeks of age. Mice were housed in an air-conditioned room (19-25?) with controlled lighting from 07:15 to 19:15 h and were given free access to food (Lab Rodent Diet No. 5002) and water. One week prior to sampling of pituitary gland, adrenalectomy was performed in mice of ADX groups (n = 12). The ADX mice received sodium chloride (0.9g/dl) in their drinking water after the surgery. All mice were killed between 08:30 and 12:30 by decapitation under isoflurane anesthesia. Brain was removed from the skull and the pituitary gland was immediately dissected. The pituitary gland was immediately frozen in liquid nitrogen, pooled together for each group, and stored at -80? until RNA extraction.

ORGANISM(S): Mus musculus

SUBMITTER: Yuichiro Nishida

PROVIDER: E-GEOD-5879 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The current study has investigated the hypothalamic gene expressions regulated by glucocorticoid (GC), key hormones in energy homeostasis. Using serial analysis of gene expression (SAGE) method, we have studied the effects of adrenalectomy (ADX) and GC on the transcriptomes of mouse hypothalamus. Approximately, 180 000 SAGE tags, which correspond to 50 000 tag species, were isolated from each group of intact or adrenalectomized mice, as well as 1, 3 and 24 hours after GC injection. ADX has upregulated diazepam binding inhibitor gene expression, while downregulating vomeronasal 1 receptor D4, genes involved in mitochondrial phosphorylation (cytochrome c oxidase 1 and NADH dehydrogenase 3), 3b-hydroxysteroid dehydrogenase-1, and prostaglandin D2 synthase. GC has increased the gene expression levels of dehydrogenase/reductase member 3, prostaglandin D2 synthase, solute carrier family 4 member 4, and five cytoskeletal proteins including myosin light chain phosphorylatable fast and troponin C2 fast. On the other hand, GC has reduced the mRNA levels of calmodilin 1 and expressed sequence tag similar to (EST) calmodilin 2, ATP synthase F0 subunit 6, and solute carrier family 4 member 3. Moreover, seven uncharacterized and 43 novel transcripts were modulated by ADX and GC. The current study has identified genes that may regulate hypothalamic systems governing energy balance in response to ADX and GC. Keywords: Hormone effect analysis Male C57BL6 mice were obtained from Charles River Laboratories (St. Constant, QC, Canada), at 12-14 weeks of age. Mice were housed in an air-conditioned room (19-25 degree) with controlled lighting from 07:15 to 19:15 h and were given free access to food (Lab Rodent Diet No. 5002) and water. One week prior to sampling of hypothalamus, adrenalectomy was performed in mice of all experimental groups (n = 12 per group). ADX mice received sodium chloride (0.9g/dl) in their drinking water after the surgery. GC (corticosterone, 0.1 mg per mouse) was subcutaneously injected to ADX mice and the hypothalamus was harvested at 1 hour (ADX + GC 1 h), 3 hours (ADX + GC 3 h) and 24 hours (ADX + GC 24 h) after the GC injection. ADX received an injection of vehicle solution (5% ethanol with 0.4% methocel A15LV premium) at 24 hours prior to sacrifice. All mice were killed between 08:30 and 12:30 by decapitation under isoflurane anesthesia. Brain was removed from the skull and the hypothalamus was immediately dissected, frozen in liquid nitrogen, pooled together for each group, and stored at -80 degree until RNA extraction.

Project description:Purpose: we sought to search for genes that are transcribed or repressed in a glucocorticoid (GC)-dependent manner in a hypothalamic region containing the paraventricular nucleus of the hypothalamus (PVH), thereby unraveling mechanisms underlying GC-induced neuroendocrine and autonomic regulation. Methods: we employed unbiased quantification of the abundance of DNA transcripts provided by RNA-seq in the PVH region of male rats and identified genes whose expression is enhanced or suppressed by the 12-day exposure to a high dose of corticosterone (Cort) by Cort implant (12-day high Cort) or deprivation of corticoids for 7 days [7-day adrenalectomy (ADX)] (Experiment 1), as well as by acute Cort increase (Experiment 2). Sham-ADX animals served as controls in Experiment 1. In Experiment 2, rats underwent ADX and supplemented with Cort in drinking water. The animals were decapitated at 1 h [acute Cort (1) 1 h] or 3 h [acute Cort (1) 3 h] following injection of HBC Cort (1 mg), a dose shown to elicit plasma Cort surge comparable to that during acute stress. A group of rats without Cort injection served as controls (ADX + Cort suppl). Differential expression was calculated as fold-changes in gene expression [measured as fragments per kilobase of exon per million reads mapped (FPKM)]. The Benjamini & Hochberg method was applied to correct the unavoidable false positive data ensued from the multiple tests, and a q-value was calculated as the false discovery rate-adjusted p-value. The q-value < 0.05 was used as the cut-off criterion. Pearson’s correlation analysis was carried out for grouping genes that showed similar expression patterns. qRT–PCR validation was performed using Power SYBR Green PCR mix. Semi-quantitative in situ hybridization was carried out using 35S-labeled cRNA probes. Results: some canonical GC target genes, among others of a broad functional spectrum, were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in Avp and Aplnr expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, 6 immediate early genes, Bsx, Egr1, Fos, Fosb, Junb, and Nr4a3, were upregulated at 1 h after Cort injection, and the changes were confirmed by qRTPCR and semi-quantitative in situ hybridization. Conclusions: the present study uncovered a broad spectrum of Cort-responsive genes, potentially involved in the regulation of the HPA-axis, as well as other neuroendocrine and pre-autonomic functions of the PVH.

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Regulation of pituitary gene expression by adrenalectomy

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