Genes associated with membrane-initiated signaling of estrogen and energy homeostasis

Abstract

During the reproductive cycle, fluctuations in circulating estrogens affect multiple homeostatic systems controlled by hypothalamic neurons. Two of these neuronal populations are arcuate proopiomelanocortin and neuropeptide Y neurons, which control energy homeostasis and feeding. Estradiol modulates these neurons either through the classical estrogen receptors (ERs) to control gene transcription or through a G protein-coupled receptor (mER) activating multiple signaling pathways. To differentiate between these two divergent ER-mediated mechanisms and their effects on homeostasis, female guinea pigs were ovariectomized and treated systemically with vehicle, estradiol benzoate (EB) or STX, a selective mER agonist, for 4 wk, starting 7 d after ovariectomy. Individual body weights were measured after each injection day for 28 d, at which time the animals were euthanized, and the arcuate nucleus was microdissected. As predicted, the body weight gain was significantly lower for EB-treated females after d 5 and for STX-treated females after d 12 compared with vehicle-treated females. Total arcuate RNA was extracted from all groups, but only the vehicle and STX-treated samples were prepared for gene microarray analysis using a custom guinea pig gene microarray. In the arcuate nucleus, 241 identified genes were significantly regulated by STX, several of which were confirmed by quantitative real-time PCR and compared with EB-treated groups. The lower weight gain of EB-treated and STX-treated females suggests that estradiol controls energy homeostasis through both ERalpha and mER-mediated mechanisms. Genes regulated by STX indicate that not only does it control neuronal excitability but also alters gene transcription via signal transduction cascades initiated from mER activation.

Figure 1

STX and estradiol significantly reduced body weight gain in ovariectomized female guinea pigs. Estradiol significantly reduced body weight gain by the fifth day after first injection. STX significantly reduced body weight gain compared with vehicle by d 12 (*, P < 0.05; **, P < 0.01). The body weights of STX-treated females were also significantly different from EB-treated females (#, P < 0.05; ##, P < 0.01). Data points represent the mean ± sem of five, five, and seven animals per group for vehicle (circles), EB (triangles), and STX (squares) treatments, respectively. Comparisons between groups were made using a two-way ANOVA (repeated measures) with post hoc Newman-Keuls paired analysis.

Figure 2

A, STX (gray) significantly (FDR P < 0.05; FC > ±1.25) regulates a number of genes compared with vehicle in the arcuate. “Up” are up-regulated genes, and “Down” are down-regulated genes. B, The percent distribution of the STX-regulated genes from the arcuate total RNA samples analyzed by microarray and divided into eight functional categories: Translation & Transcription; Channels & and Membrane Proteins; Signaling Molecules; Cell Cycle & Growth; Metabolism and Mitochondria; Protein Trafficking & Catabolism; Cell Communication; and Unknown. The cDNA microarray chip was created from a brain-specific, estrogen-regulated guinea pig cDNA library produced using SSH.

Figure 3

A representation of the percentage of the STX-regulated genes within each of the four P value ranges. All 241 genes with a FC more than ±1.25 were divided into four categories based on their associated P value for the FDR. These categories are: P < 0.05 but more than 0.01; P < 0.01 but more than 0.001; P < 0.001 but more than 0.0001; and P < 0.0001.

Figure 4

qPCR confirms regulation of multiple genes by STX in the arcuate nucleus. The relative mRNA expression for selected control genes (A and B) and selected genes of interest (C and D) from microdissected arcuate tissue from vehicle-treated [propylene glycol (n = 5) or oil (n = 7); black bars] females and STX- (n = 7) or EB-treated (n = 6) females (gray bars). The expression values were calculated using the ΔΔCT method where the calibrator was the average ΔCT of the vehicle-treated samples. Bar graphs represent the mean ± sem. Statistics: two-tailed Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with vehicle).

Figure 5

A, Classical ER membrane-associated and nuclear-associated signaling. B, Signaling pathways for the novel membrane ER (mER). 1) Estrogen can activate the classical ER-mediated (ERα/β) pathways leading to changes in mRNA transcription of relevant genes via ERE. 2) Both ERs are regulated by STX, and may be a mechanism for the interaction of classical ER and mER signaling. 3) Estrogen can bind to membrane-associated classical ER leading to the activation of signal transduction pathways to control gene transcription and cellular functions. The putative mER is a G protein-coupled receptor (GPCR) linked to G_q/11 protein that activates PLC-PKC-PKA pathway may control gene transcription via pCREB-CRE. The activation of PKC can also lead to changes in gene transcription via the phosphorylation of other signaling proteins (MAPK/ERK). 4) The hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP₂) into 1,4,5-trisphosphate (IP3) may also initiate calcium-dependent signaling molecules (CaM/CaMK) to control transcription. Several of these genes are associated with cation and anion channels (Ca_v3.1, GLR) or proteins that impinge on channel functions (AKAP). 5) Changes in neuronal excitability either by channel expression or channel modulation may also control gene expression. A few of the genes analyzed by qPCR are involved in calcium signaling (CaMK II) and protein trafficking (GEC1).