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Effect of AMPK activation by AICAR on MA-10 Leydig cell transcriptome

ABSTRACT: Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production.

ORGANISM(S): Mus musculus

PROVIDER: GSE50118 | GEO | 2014/08/19

SECONDARY ACCESSION(S): PRJNA216106

REPOSITORIES: GEO

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Project description:Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. MA-10 Leydig cells were treated with either DMSO (control), 10 uM forskolin or forskolin+Aicar (1 mM) for 1.5 h before total RNA extraction

Project description:Steroidogenic acute regulatory protein (Star) facilitates cholesterol transfer into the inner mitochondrial membrane in the acute and regulated production of steroid hormones. Mice lacking Star (Star-/-) share the phenotypes with patients with congenital lipoid adrenal hyperplasia such as compromised production of steroid hormones and florid accumulation of cholesterol esters in adrenal glands and gonads. To define specific patterns of molecular changes with disruption of Star, we performed a transcriptome analysis of steroidogenic cells in adrenal glands. We harvested adrenal glands at E17.5 or 18.5 from seven wild-type (Star+/+) mice or four Star-/- mice having the transgene targeting enhanced green fluorescent protein (eGFP) under the control of regulatory sequences of mouse Star gene. Steroidogenic cells were selectively isolated by fluorescent-activated cell sorting. The gene expression profile of the fluorescence-positive cells was obtained with Agilent Whole Mouse Genome Microarray and was confirmed by quantitative real-time PCR. We identified 961 and 622 genes that were significantly up-regulated and down-regulated, respectively, in Star-/- mice compared with Star+/+ mice (fold difference ≥2, p value of Student t test <0.05, and Benjamini-Hochberg false discovery rate of multiple comparison test <0.2). In Star-/- mice, expression levels of genes involved in cholesterol mobilization and efflux or immune response as antigen presenting cells were significantly increased, and transition from fetal to adult adrenocortical cells were significantly decreased, whereas those of genes related to steroid hormone biosynthesis or cholesterol biosynthesis and influx were not significantly changed. The trancriptome analysis revealed hitherto undescribed characteristic changes in adrenocortical cells and expanded our understanding of the pathophysiology of the steroidogenic cells with disruption of Star. Gene expression profiles of isolated adrenal steroidogenic cells were compared between seven wild-type mice and four knockout mice lacking steroidogenic acute regulatory protein at E17.5-18.5.

Project description:Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cells function may have ultimate consequences. 3-methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and luteinizing hormone (LH)-stimulated Leydig cells following 48 h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10µM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 7540 proteins were identified in which the abundance of 87 proteins in unstimulated Leydig cells and 146 proteins in LH-stimulated Leydig cells were found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO2-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathion-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO2-DDE induced endocrine disruption in Leydig cells.

Project description:Ovarian granulosa cells play a central role in steroidogenesis, which is critical for female reproduction. Follicle-stimulating hormone (FSH) promotes cAMPâmediated signaling to regulate granulosa cell steroidogenesis. We have shown previously that 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE) inhibits FSH- and dibutyryl cAMP-stimulated steroidogenesis, and affects the mRNA levels of steroidogenic pathway enzymes in rat granulosa cells. However, HPTE showed a differential effect in FSH- and cAMP-stimulated cells in that HPTE more completely blocked FSH- when compared to cAMP-driven steroidogenesis. The objective of this study was to analyze the effects of HPTE on global gene expression profiles in untreated granulosa cells and those challenged with FSH or cAMP. Granulosa cells from immature rats were cultured with 0, 1, 5, or 10 ÂµM HPTE in the presence and absence of either 3 ng FSH/ml or 1 mM cAMP for 48 h. Total RNA was isolated for microarray analysis using the GeneChip Rat Genome 230 2.0 and ArrayAssist Microarray Suite. An investigation of changes in gene expression across all HPTE treatments showed that HPTE altered more genes in FSH- (~670 genes) than in cAMP-stimulated cells (~366 genes). Analysis confirmed that HPTE more effectively inhibited FSH- than cAMP-induced steroid pathway gene expression and steroidogenesis. Furthermore, expression patterns of novel genes regulating signal transduction, transport, cell cycle, adhesion, differentiation, motility and growth, apoptosis, development, and metabolism were all altered by HPTE. This study further established that HPTE exerts differential effects within the granulosa cell steroidogenic pathway, and revealed that these effects include broader changes in gene expression. Experiment Overall Design: Ovaries were dissected from immature (21-27 days old), Sprague-Dawley rats and were placed into ice cold Hamâs F12 medium. Granulosa cells were isolated from ovaries free of fat pads and surrounding connective tissue by performing non-enzymatic needle puncture method . Following two washes by centrifugation at 1400rpm/250xg for 5 min at 4 CË, the cells were plated and cultured at approximately 3-4x105 viable cells/mL/well in DMEM/F-12 medium containing 5% FCS at 37 CË in 5% CO2 in a 24-well culture plate for 24 hours. The cells were then cultured in serum-free DMEM/F12 containing 0.1 Î¼M androstenedione as a substrate for aromatization into E2. Experiment Overall Design: Cells were treated in triplicate with increasing doses of HPTE (0, 1, 5, and 10 ÂµM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Similar doses of HPTE were previously used (Zachow and Uzumcu, 2006). Cultures were terminated at 48 h following the addition of treatments. Experiment Overall Design: Cell lysate was collected for total RNA isolation for DNA microarray analysis. Total RNA were extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA qualities were assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis. Forty nanograms of total RNA from each sample was used to generate a high fidelity cDNA for array hybridization using NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen, Cat#2300-60). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturerâs protocol. The detection and quantization of target hybridization was performed with an Affymetrix GeneChip Scanner. Data were assessed for array performance prior to analysis and analyzed using ArrayAssist (Stratagene).

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Effect of AMPK activation by AICAR on MA-10 Leydig cell transcriptome

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