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Physiol. Genomics 25: 96-104, 2006. First published December 20, 2005; doi:10.1152/physiolgenomics.00232.2005
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Received 19 September 2005; accepted in final form 15 December 2005.
Physiological Genomics 25:96-104 (2006)
American Physiological Society © 2006 American Physiological Society

Regulation of hypothalamic gene expression by glucocorticoid: implications for energy homeostasis

Yuichiro Nishida, Mayumi Yoshioka and Jonny St-Amand

Functional Genomics Laboratory, Molecular Endocrinology and Oncology Research Center, Laval University Medical Center (CHUL), and Department of Anatomy and Physiology, Laval University, Quebec City, Quebec, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study investigated the hypothalamic gene expressions regulated by glucocorticoids (GC), key hormones in energy homeostasis. Using the serial analysis of gene expression (SAGE) method, we 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 h after GC injection. ADX 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), 3ß-hydroxysteroid dehydrogenase-1, and prostaglandin D2 synthase. GC 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 reduced the mRNA levels of calmodulin 1 and expressed sequence tag similar to calmodulin 2, ATP synthase F0 subunit 6, and solute carrier family 4 member 3. Moreover, 7 uncharacterized and 43 novel transcripts were modulated by ADX and GC. The present study has identified genes that may regulate hypothalamic systems governing energy balance in response to ADX and GC.

transcriptome; serial analysis of gene expression; adrenalectomy; obesity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CLINICAL FEATURES OF ANOREXIA and weight loss in patients with adrenal insufficiency as well as increased food intake and weight gain in patients with glucocorticoid (GC) excess implicate adrenal GC in energy homeostasis (18). Obesity is dependent on GC action, because rodent models of obesity with pathologies of genetic (13), dietary (65), and hypothalamic (38) origins are normalized by adrenalectomy (ADX) and restored by GC replacement (27). In normal rodents and humans, GC increases food intake and body weight, as well as inducing metabolic features of obesity, such as insulin resistance, hyperinsulinemia, hypertriglyceridemia, and hyperleptinemia (30, 76). Interestingly, an administration of leptin, which barely affects body weight and food intake, becomes powerful and long-lasting in ADX rats, suggesting that GC antagonizes the central action of leptin (75). GC not only increases food intake but also decreases energy expenditure by suppressing thermogenesis in mouse brown adipose tissue (63). Thus GC promotes positive energy balance, whereas a lack of GC is linked to hypophagia and reduced body weight.

The hypothalamus is a brain center that regulates energy homeostasis by integrating peripheral signals such as leptin, insulin, and GC. Thus the effects of ADX and GC on hypothalamic gene expression have been a major focus of many previous studies (45, 54, 60, 73). However, no study has previously investigated effects of ADX and GC in the transcriptome of the hypothalamus. The serial analysis gene expression (SAGE) method accurately measures the expression levels of tens of thousands of genes, previously known or not, and finds the genes related to diseases or the effects of stimuli (59, 68). Although other techniques such as DNA microarray are limited by their ability to analyze only previously known transcripts, SAGE does not require a priori knowledge of the sequence of mRNA transcripts expressed in the tissues of interest. In addition, we recently showed (22) that the SAGE method has a very high reproducibility, with r2 = 0.96. Moreover, no mRNA species had significant difference in their level of expression estimated by two SAGE libraries constructed from the same pool of total RNA (22). Thus, in the present study, we intended to identify potential mediators for the effects of GC on hypothalamic systems governing energy balance by using the SAGE strategy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rodents and hypothalamus sampling.
The protocols were approved by the Laval University Committee for Animal Protection. Male C57BL/6 mice were obtained from Charles River Laboratories (St. Constant, QC, Canada), at 12–14 wk of age. Mice were housed in an air-conditioned room (19–25°C) with controlled lighting from 0715 to 1915 and were given free access to food (Lab Rodent Diet no. 5002) and water.

One week before sampling of hypothalamus, ADX was performed in mice of all experimental groups (n = 12 per group) except for the intact group (n = 51). ADX mice received sodium chloride (0.9 g/dl) in their drinking water after the surgery. GC (corticosterone, 0.1 mg per mouse) was subcutaneously injected into ADX mice, and the hypothalamus was harvested at 1 (ADX + GC 1 h, n = 12), 3 (ADX + GC 3 h, n = 12), and 24 (ADX + GC 24 h, n = 12) h after the GC injection. The physiological dose of GC was determined on the basis of previous studies in our research center (16). ADX mice received an injection of vehicle solution (5% ethanol with 0.4% methocel A15LV premium) at 24 h before death. All mice were killed between 0830 and 1230 by decapitation under isoflurane anesthesia. The brain was removed from the skull, and the hypothalamus was immediately dissected, following the boundaries described by Paxinos and Franklin (53). The whole hypothalamus was taken, using the optic chiasm as the rostal limit and the mammillary bodies as caudal reference. The hypothalamus was immediately frozen in liquid nitrogen, pooled together for each group, and stored at –80°C until RNA extraction. All mice were handled in a facility approved by the Canadian Council on Animal Care in accordance with their Guide for Care and Use of Experimental Animals.

SAGE and data analysis.
Total RNA was isolated from tissues with a RNA extraction kit (TRIzol reagent; Invitrogen Canada, Burlington, ON, Canada). Approximately 6 µg of mRNA was extracted with an Oligotex mRNA Mini Kit (Qiagen, Mississauga, ON, Canada). The SAGE method was performed as previously described (59, 68). In brief, double-strand cDNA was synthesized from the mRNA with a biotinylated (T)18 primer and a cDNA synthesis kit (Invitrogen Canada). The cDNA libraries were digested with the restriction enzyme NlaIII (New England Biolabs, Pickering, ON, Canada), which recognizes CATG sequences. The 3'-terminal cDNA fragments were captured with streptavidin-coated magnetic beads (Dynal, Biotech, Brown Deer, WI). After ligation of two annealed linker pairs to the NlaIII-compatible sticky ends, the cDNA fragments were digested with the tagging enzyme BsmFI (New England Biolabs), thereby releasing cDNA fragments including the short 15-bp tags. A blunting kit (Takara Bio, Otsu, Japan) was used for the blunting and ligation of the two populations of cDNA fragments, and the ligation products were amplified by PCR (59). The PCR products were digested with NlaIII and the band containing the ditags was extracted from the 12% polyacrylamide gel. The purified ditags were self-ligated to form concatemers with T4 ligase (Invitrogen Canada). The concatemers were cloned into SphI site of pUC19. White colonies were screened by PCR and agarose gel to select long inserts for automated sequencing. The sequence and occurrence of each tag were analyzed by the software SAGEana program, which is a new version of SAGEparser.pl (22). Tags corresponding to linker sequences were discarded, and duplicate concatemers were counted only once. To identify the transcripts, the sequences of 15-bp SAGE tags (NlaIII site CATG plus adjacent 11-bp tags) were matched with public databases. To avoid the possibility of sequencing errors in the expressed sequence tag (EST) database, we did not consider the matches that were identified only once among the numerous sequences of a UniGene cluster. A minimum of one EST with a known polyA tail had to be in the UniGene cluster to identify the last NlaIII site on the corresponding mRNA. The tag numbers were normalized by 100,000. The transcripts were classified according to their functions based on the genome directory (1) and other literature. The SAGE tags that did not match any sequences in the public databases were classified as novel transcripts.

Quantitative real-time PCR.
Two mRNAs, prostaglandin D2 synthase (Mm. 1008, NM_008963) and solute carrier family 4 member 3 (Mm. 5053, NM_009208), were also measured by quantitative (Q)RT-PCR. The RNA samples pooled together for each group, ADX (n = 12), ADX + GC 1 h (n = 12), ADX + GC 3 h (n = 12), and ADX + GC 24 h (n = 12), were used. First-strand cDNA was synthesized with 5 µg of isolated RNA in a reaction containing 200 U of Superscript III RNase H-RT (Invitrogen Life Technologies, Carlsbad, CA), 300 ng of oligo(dT)18, 500 mM deoxynucleotide triphosphates, 5 mM dithiothreitol, and 34 U of human RNase inhibitor (Amersham Pharmacia, Piscataway, NJ) in a final volume of 50 µl. The reaction was performed at 50°C for 2 h and then treated with RNase A for 30 min at 37°C. The resulting products were purified with Qiaquick PCR purification kits (Qiagen, Valencia, CA). The cDNA corresponding to 20 ng of total RNA was used to perform fluorescence-based real-time PCR quantification with the LightCycler real-time PCR apparatus (Roche, Nutley, NJ). Reagents were obtained from the same company and were used as described by the manufacturer. The conditions for PCR reactions were denaturation at 95°C for 10 s, annealing at 56–66°C for 5 s, and elongation at 72°C for 7–13 s. The reaction was then heated for 3 s at 2°C lower than the melting temperature of the DNA fragment. The reading of the fluorescence signal was taken at the end of the heating to avoid nonspecific signal. A melting curve was performed to assess nonspecific signal. Oligoprimer pairs that allow the amplification of ~200 bp were designed by GeneTools software (Biotools, Edmonton, AB, Canada) and their specificity was verified by BLAST in the GenBank database. Data calculation and normalization were performed with the second derivative and double correction method, using the housekeeping gene hypoxanthine guanine phosphoribosyl transferase 1 (44). Hypoxanthine guanine phosphoribosyl transferase 1 has stable expression levels from embryonic life through adulthood in various tissues (71). The expression levels of mRNA were expressed as number of copies per microgram of total RNA, using a standard curve of crossing point vs. logarithm of the quantity. The standard curve was established with known cDNA amounts of 0, 102, 103, 104, 105, and 106 copies of hypoxanthine guanine phosphoribosyl transferase 1 and a LightCycler 3.5 program provided by the manufacturer (Roche). The QRT-PCR was performed in duplicate and had <2.9% standard deviation.

Statistical analysis.
To detect the effects of ADX and GC on mRNA levels with more than a twofold change, a comparative count display test was used (41). Differences were considered to be statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Number of tags analyzed and transcripts differentially expressed by ADX and GC.
Five libraries (intact, ADX, and ADX + GC 1 h, 3 h, and 24 h) were generated to identify hypothalamic mRNAs regulated by ADX and GC. Approximately 180,000 SAGE tags, which correspond to 50,000 tag species, were sequenced in each group (Table 1). In total, 71 transcripts were differentially expressed by ADX and GC administration. Twenty-one well-characterized transcripts differentially expressed at a significant level (P < 0.05) are presented in Tables 2Go–4, according to their functions. Among these, 5 are involved in cell signaling and communication (Table 2), 11 are involved in metabolism (Table 3), and the other 5 are involved in the cytoskeleton (Table 4). Seven uncharacterized and forty-three novel transcripts differentially expressed are presented in Tables 5 and 6, respectively.


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  		Table 1. Number of tags and tag species analyzed in intact, adrenalectomized, and glucocorticoid treated mice

 

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  		Table 2. ADX- and GC-regulated transcripts involved in cell signaling and communication

 

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  		Table 3. Differentially expressed transcripts involved in metabolism

 

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  		Table 4. Differentially expressed transcripts involved in cytoskeleton

 

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  		Table 5. Differentially expressed transcripts with uncharacterized functions

 

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  		Table 6. Novel transcripts differetially expressed

 
Differentially expressed transcripts involved in cell signaling and communication.
ADX and GC modulated the expression of five genes involved in cell signaling and communication (Table 2). ADX upregulated the mRNA level of diazepam binding inhibitor by 2.9-fold, while downregulating vomeronasal 1 receptor D4 by 4.4-fold. GC induced gene expression of dehydrogenase/reductase member 3 at 24 h after administration, while suppressing calmodulin 1 and EST calmodulin 2 at 1 and 24 h.

Differentially expressed transcripts involved in metabolism.
Eleven transcripts involved in metabolism were regulated by ADX and GC in the hypothalamus (Table 3). GC injection caused a biphasic decrease in ATP synthase F0 subunit 6 mRNA. ADX drastically reduced the expression levels of genes involved in mitochondrial phosphorylation, such as three transcript species of cytochrome-c oxidase 1 and NADH dehydrogenase 3. For lipid metabolism, two prostaglandin D2 synthase mRNAs with the SAGE tags CATG GTGACCTGGCC and CATG GCCACCCTCTA were downregulated, whereas GC induced gene expression of another isoform with a tag sequence CATG GTAAGCGCTAC by sixfold. In addition, ADX downregulated gene expression of 3ß-hydroxysteroid dehydrogenase-1. For transport function, GC downregulated the mRNA level of solute carrier family 4 member 3 at 3 h, whereas GC upregulated member 4 in this family at 24 h.

Differentially expressed transcripts involved in cytoskeleton.
GC induced the expression of five genes encoding motor proteins, such as myosin heavy polypeptide 4, myosin light chain phosphorylatable fast, myosin light polypeptide 1, troponin C2 fast, and troponin I skeletal fast 2 (Table 4). These increases occurred slowly at 24 h after GC treatment.

Uncharacterized and novel transcripts differentially expressed.
Seven uncharacterized transcripts were regulated by ADX and GC (Table 5). Among these, four were modulated (up- and/or downregulation) by GC, whereas the other three were upregulated by ADX. Moreover, 43 novel transcripts differentially expressed by ADX and GC were found (Table 6). Among these, two novel transcripts with tag sequences CATG AAAAATCATCG and CATG TCATTGGTCGC were significantly downregulated by ADX and reversed by GC. GC also upregulated three novel transcripts with tag sequences CATG ACCCAGAGGGC, CATG GCTGCCCTCCT, and CATG AGCTTGGCCTG. ADX upregulated 3 novel transcripts by 7- to 11-fold, while as many as 35 novel transcripts were downregulated by ADX.

Confirmation of SAGE data by QRT-PCR.
As shown in Fig. 1, the changes in the expression levels of prostaglandin D2 synthase and solute carrier family 4 member 3 measured by QRT-PCR were similar to the changes measured by SAGE.


Figure 1
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  		Fig. 1. Changes in the expression levels of prostaglandin D2 synthase (Mm. 1008, NM_008963; A) and solute carrier family 4 member 3 (Mm. 5053, NM_009208; B) measured by serial analysis of gene expression (SAGE; {circ}) and quantitative (Q)RT-PCR (•).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Differentially expressed transcripts involved in cell signaling and communication.
The present study has detected an upregulation of diazepam binding inhibitor gene expression in ADX. Octadecaneuropeptide is generated by the proteolytic cleavage of the diazepam binding inhibitor, and it has the ability to displace benzodiazepine diazepam from its membrane receptors (29). Interestingly, central administration of the octadecaneuropeptide markedly inhibited food intake in both food-deprived and normally fed rodents (21). Furthermore, acute octadecaneuropeptide administration increased mRNA level of the anorexigenic proopiomelanocortin (POMC) and decreased mRNA expression of neuropeptide Y in the arcuate nucleus (15). The diazepam binding inhibitor gene is strongly expressed around the region of the third ventricle and arcuate nucleus of the hypothalamus (24, 64). Thus the upregulation of diazepam binding inhibitor gene suggests its important role in weight reduction in ADX. In addition, the change in the expression level of this gene could also influence the hypothalamic regulation of neuroendocrine factors in ADX. The administration of GC decreases prolactin gene expression in the pituitary gland, whereas ADX and central injection of octadecaneuropeptide both increase the expression level of prolactin gene (70). Prolactin is well known to stimulate lactation in female mammalians. In male pituitary gland, prolactin gene is expressed at high levels (50), and it may have important roles for normal testicular function (23).

GC reduced the mRNA levels of calmodulin 1 and EST calmodulin 2 in the hypothalamus. Calmodulin (CaM), an intracellular Ca2+ binding protein, is abundantly expressed in the brain (77), and CaM-dependent phosphorylation and dephosphorylation regulate the synthesis and release of several neurotransmitters (36). A previous study showed that leptin injection increases gene expression of calcineurin, a CaM-dependent phosphatase, by approximately twofold in arcuate nucleus of the hypothalamus, suggesting that CaM and Ca2+/CaM play a role in the hypothalamic leptin signaling (49). Because GC is known to cause leptin resistance in the hypothalamus (75), the downregulation of mRNA levels of calmodulin 1 and EST calmodulin 2 by GC might suggest its involvement in GC-induced leptin resistance.

Vomeronasal receptor gene families are expressed in vomeronasal sensory neurons of the vomeronasal organ in the nasal cavity (52). Seven-transmembrane-domain proteins encoded by the vomeronasal receptor genes are believed to be pheromone receptors that detect pheromones, chemical signals that modulate reproductive, defensive, and ingestive behavior as well as neuroendocrine secretion (20, 37). A recent study revealed that there is an extreme degree of diversity in the vomeronasal receptor 1 repertoire (293 vomeronasal receptor 1 genes) in the mouse genome (55). A human vomeronasal receptor 1 homolog, vomeronasal 1 receptor-like gene, has also been identified (55). Because only a handful of chemical compounds with pheromonal effects have been isolated from the mouse, the unusually high diversity in the vomeronasal receptor 1 gene superfamily suggests nonpheromonal functions of some vomeronasal receptor 1 (55). Although there is no previous report on the role of this gene in the hypothalamus, the present result has shown that vomeronasal receptor 1 D4 is expressed at middle level in the intact hypothalamus, and its expression level reduces to one-fourth by removal of GC. Thus the result supports the idea that vomeronasal receptor 1 D4 has nonpheromonal functions. These functions would be involved in hypothalamic regulations of instinct behaviors and homeostasis, such as feeding and sexual behaviors as well as temperature control and neuroendocrine secretion.

Short-chain dehydrogenase/reductase member 3, also known as retinal short-chain dehydrogenase/reductase 1, regenerates all-trans-retinol (vitamin A) from all-trans-retinal (vitamin A aldehyde) in the visual cycle in the retina of insects (31). On the other hand, in human neuroblastoma cell lines short-chain dehydrogenase/reductase member 3 induces the accumulation of retinyl esters (retinyl palmitate), which can be interpreted as the attempt of cells to accumulate local retinol storage in the case of retinol availability (14). Several previous reports and the present results suggest an important role of retinoid metabolism in the hypothalamus for energy homeostasis. To date, the localization of short-chain dehydrogenase/reductase member 3 in the hypothalamus has not been clarified. However, the mRNA and protein of retinoid X receptors (receptors for 9-cis-retinoic acid) have been reported to be strongly expressed in arcuate, dorsomedial, and ventromedial nuclei in the hypothalamus (40). Retinoid X receptor-{gamma}-deficient mice have significantly higher serum T4 and TSH levels and increased metabolic rate than wild-type controls, but the weights of these animals are not significantly different (8). In addition, another recent study showed that retinoid X receptor-{gamma}-deficient mice are resistant to gain in fat mass in response to high-fat feeding (33). Thus short-chain dehydrogenase/reductase member 3 could be an important molecule that modulates the hypothalamic retinoid metabolism and energy homeostasis, because the present study has identified an increased gene expression of short-chain dehydrogenase/reductase member 3 as a potential regulator that may induce positive energy balance.

Differentially expressed transcripts involved in metabolism.
The first rapid decrease of the ATP synthase F0 subunit 6 mRNA level occurred at 1 h, and the second decrease was observed at 24 h after GC treatment. Recent studies showed that hypothalamic energy status plays an important role in feeding behavior (11, 42). AMP-activated protein kinase (AMPK), which is activated by an increase in the ratio of AMP to ATP within the cell, is the central component of a protein cascade that plays a key role in the regulation of energy control (11). Interestingly, in the hypothalamus, orexigenic ghrelin activates AMPK, whereas satiety signals of leptin and insulin inhibit AMPK (11). In addition, pharmacological (5-aminoimidazole-4-carboxamide ribose) activation (phosphorylation) of AMPK in the hypothalamus increases food intake (11). It is noteworthy that ATP synthesis inhibitor, which generates a low cellular ATP concentration, activates AMPK (42). Thus reduction of ATP synthase F0 subunit 6 mRNA level by GC may contribute to the activation of AMPK.

The most dramatic effect of ADX was the decrease in the levels of mRNAs involved in mitochondrial phosphorylation, such as the three transcript species of cytochrome-c oxidase 1 and NADH dehydrogenase 3, suggesting a reduction of mitochondria after ADX. Critical roles of mitochondrial fatty acid oxidation in the hypothalamus for the regulation of food intake have been shown (43, 51). Indeed, intracerebroventricular treatment of mice with fatty acid synthase (FAS) inhibitors led to inhibition of feeding and dramatic weight loss (43). In addition, inhibition of hypothalamic lipid oxidation by decreasing the activity of carnitine palmitoyltransferase-1, which regulates long-chain fatty acid (LCFA) entry into mitochondria, where the LCFAs undergo ß-oxidation, substantially diminished food intake (51). If the amount of mitochondria in the hypothalamus was reduced by ADX, as suggested by the present results, this might reduce ß-oxidation and food intake in ADX. Future studies are needed to determine the biological roles of reduction in the hypothalamic mitochondrial enzymes such as cytochrome-c oxidase 1 and NADH dehydrogenase 3 in ADX.

Prostaglandin D2 is considered as an endogenous sleep-promoting factor. Prostaglandin D2 synthase is an enzyme that produces prostaglandin D2 in the brain (66). Using Northern and Western blot assays, a previous study also showed that the synthetic glucocorticoid dexamethasone increases mRNA and protein levels of prostaglandin D2 synthase in a mouse hypothalamic neuronal cell line (28). The present result is in agreement with a previous in vitro study and is the first in vivo evidence that GC induces hypothalamic gene expression of prostaglandin D2 synthase. Interestingly, the inhibitor of prostaglandin D2 synthase reduced food intake as well as sleep (61). Prostaglandin D2 synthase is expressed in the arcuate and ventromedial nuclei of medial basal hypothalamus (48). This subarea in the hypothalamus is pivotal in the hypothalamic control of energy homeostasis (35). Thus the present and previous results may suggest an important implication of this enzyme as a regulator for GC-induced positive energy balance. In addition, the modulation of prostaglandin D2 synthase gene by GC can also influence hypothalamic neuroendocrine function. The intraventricular administration of prostaglandin D2 inhibits luteinizing hormone secretion (39). Consistently, GC is known to suppress gene expression of gonadotropin-releasing hormone (5) and inhibit testicular steroidogenesis (4). Thus the increased hypothalamic gene transcription of prostaglandin D2 synthase suggests its critical role in the reduction of reproductive hormones by GC.

ADX increases binding affinities of GABA receptors for a GABA type A (GABAA) receptor agonist, muscimol, by 38% in the hypothalamus (46), thus suggesting that ADX would enhance GABA action. GABA, acting on GABAA receptors, inhibits activity of several key steroidogenic enzymes, including 3ß-hydroxysteroid dehydrogenase-1 in the hypothalamus (25). Indeed, it was shown in the frog hypothalamus that most of the 3ß-hydroxysteroid dehydrogenase-1-positive neurons located in the suprachiasmatic, dorsal, and ventral hypothalamic nuclei also contain GABAA receptors (25). Thus the present result that ADX downregulated 3ß-hydroxysteroid dehydrogenase-1 gene expression is consistent with these previous studies. The fact that subpopulations of both orexigenic neuropeptide Y neurons (17) and anorexigenic POMC neurons (34) in the hypothalamus release GABA may also suggest an involvement of 3ß-hydroxysteroid dehydrogenase-1 in energy homeostasis. Because GC injection did not restore the effect of ADX, there is a possibility that this gene might be regulated by adrenal factors other than GC, including mineralocorticoid, epinephrine, and norepinephrine. Thus the present results help to explore the biological role of this gene in the hypothalamus.

Intracellular pH can change rapidly and transiently in response to neuronal activation by hormones, as well as transmitters, growth factors, and other messengers (19). The change in intracellular pH, in turn, has an impact on central nervous system function via synaptic transmission, neuronal excitability, and metabolic enzyme activity (19). Thus the modulation of intracellular pH appears to be crucial for proper neural function. Differentially expressed genes in the present study, solute carrier family 4 member 3 and member 4, are key players in intracellular pH regulation in the central nervous system (19, 74). Although there are previous reports that showed an importance of potassium channels in hypothalamic neurons, which are opened by leptin (57) and insulin (58), to our knowledge, no previous study has investigated the role of ion exchangers in the hypothalamus. Solute carrier family 4 member 3 is an acid loader that exchanges extracellular chloride for intracellular bicarbonate and is responsible for the intracellular pH recovery from alkaline loads (19, 74). On the other hand, solute carrier family 4 member 4 is an acid extruder that exchanges extracellular bicarbonate for intracellular sodium and participates in the intracellular pH recovery from acid loads (56, 74). Thus the present results suggest that GC may lead to an intracellular alkalization by reducing the mRNA level of an acid loader (solute carrier family 4 member 3) and by inducing gene expression of an acid extruder (solute carrier family 4 member 4). Although solute carrier family 4 members 3 and 4 are known as key players in intracellular pH regulation in the central nervous system (19, 74), to our knowledge there are no previous reports on the localization of these mRNAs in the hypothalamus. Thus the localization of these transcripts in the hypothalamus and the physiological role of intracellular pH modulation by these ion exchangers in hypothalamic control of energy homeostasis have arisen as an intriguing subject to be clarified.

Differentially expressed transcripts involved in cytoskeleton.
The GC has modulated levels of mRNAs involved in the cytoskeleton, such as myosin heavy polypeptide 4, myosin light polypeptide 1, troponin C2 fast, and troponin I skeletal fast 2 at 24 h after the treatment, with dramatic increases by 6- to 15.5-fold. Myosins are motor proteins that move on filamentous actin (F-actin), using the hydrolysis of ATP, transporting vesicles, organelles, protein complexes, and mRNA, to various sites in the cell (47). In neurons, multiple myosins have been found (1a, 7). Myosin VI plays a role in the endocytosis of glutamate receptors, and myosin VI-deficient neurons have fewer synapses and dendritic spines (10, 32). In addition, troponin C slow mRNA has been reported to be expressed at high levels in brain (6). However, the functions of myosin and troponin in brain are largely unknown.

Several investigators have proposed that in secretory cells a network of F-actin underlies the plasma membrane and regulates exocytosis by acting as a barrier that impedes the apposition of secretory granules to membrane fusion sites, which is called the "barrier theory" (2, 9). Interestingly, GC stabilizes F-actin filaments in diverse cell types including At-T-20 corticotrophs, and the stabilization of F-actin correlates with inhibition of ACTH secretion in the corticotrophs; thus it is considered to be a possible mechanism for a negative feedback on GC to the pituitary gland (12). The stabilization of F-actin filaments is accompanied by increased expression of the actin-binding protein caldesmon, which regulates the interaction among actin, tropomyosin, and myosin (12, 67). Some evidence even implicates myosin in the polymerization of actin (12). Thus genes encoding actin-binding proteins such as myosin (heavy polypeptide 4 and light polypeptide 1) and troponin (C2 fast and I skeletal fast 2), which were induced by GC in the present study, may be candidate molecules involved in the negative feedback system on GC at the hypothalamic level.

Expression levels of appetite and satiety genes known to be regulated by ADX and GC.
In normal rodents, removal of GC by ADX significantly reduces hypothalamic mRNA levels of orexigenic signals such as neuropeptide Y (45), melanin-concentrating hormone, and orexin (26). On the other hand, anorexigenic genes such as interleukin-1ß, leptin receptor, and signal transducers and activators transcription-3 are induced by ADX (45). The changes of neuropeptide Y and interleukin-1ß mRNAs by ADX are reversed by GC administration (45). In addition, GC is well known to negatively feed back on hypothalamic corticotropin-releasing hormone expression (72). We could not detect tags corresponding to neuropeptide Y (Mm. 154796, NM_023456), melanin-concentrating hormone (Mm. 179378, NM_029971), interleukin-1ß (Mm. 222830, NM_008361), signal transducers and activators transcription-3 (Mm. 249934, NM_213659), and corticotropin-releasing hormone (Mm. 290689, NM_205769), probably because of their low abundance, because they have at least one CATG in their mRNA sequences. In the present study, orexin mRNA (Mm .10096, NM_010410) level did not change significantly with ADX [intact: 33 tags, ADX: 76 tags; not significant (NS)] or with GC (ADX: 76 tags, GC 1 h: 95 tags, GC 3 h: 82 tags, GC 24 h: 98 tags; NS). Using in situ hybridization, a previous study showed a reduction in cocaine- and amphetamine-regulated transcript (CART) mRNA levels in the paraventricular and arcuate nuclei in ADX rats, which was restored on dexamethasone treatment but not by subcutaneous GC pellets (69). In the present study, CART mRNA (Mm. 75498, NM_013732) levels did not change significantly (intact: 37 tags, ADX: 31 tags, GC 1 h: 36 tags, GC 3 h: 31 tags, GC 24 h: 42 tags; NS). It is important to recognize that the present data were obtained from whole hypothalamus. The discrepancy could be due to different responses of CART mRNA level to GC in different hypothalamic nuclei, because ADX lowered CART-positive cells in paraventricular nuclei but there were no significant changes in the supraoptic nucleus (3). POMC (Mm. 277996, M30489) and agouti-related protein (AGRP) expressions were also unaltered by ADX and GC [POMC intact: 0 tags, ADX: 1 tag, GC 1 h: 0 tags, GC 3 h: 0 tags, GC 24 h: 0 tags (NS); AGRP intact: 1 tag, ADX: 0 tags, GC 1 h: 1 tag, GC 3 h: 3 tags, GC 24 h: 2 tags (NS)]. These results are in agreement with a previous study that used whole hypothalamus (73).

Uncharacterized and novel transcripts differentially expressed.
The present study targeted not only well-characterized transcripts but also uncharacterized and novel transcripts differentially expressed by ADX and GC. Indeed, 7 uncharacterized and 43 novel transcripts differentially expressed by ADX and GC were found. Further characterization of these uncharacterized and novel transcripts would contribute to understanding of multiple physiological functions of the hypothalamus, including satiety, temperature control, and energy homeostasis as well as neuroendocrine regulation, mood, and numerous autonomic functions.

In conclusion, the present study has for the first time investigated the effects of GC, key hormones involved in energy homeostasis, on global gene expressions in the hypothalamus, which is a brain center regulating whole body energy homeostasis. ADX and GC regulate hypothalamic mRNAs in three general functions, such as cell signaling and communication (diazepam binding inhibitor, calmodulin 1, EST calmodulin 2, dehydrogenase/reductase member 3, and vomeronasal 1 receptor D4), metabolism (ATP synthase F0 subunit 6, cytochrome-c oxidase 1, NADH dehydrogenase 3, 3ß-hydroxysteroid dehydrogenase-1, prostaglandin D2 synthase, and solute carrier family 4 member 3 and 4), and the cytoskeleton (myosin heavy polypeptide 4, myosin light chain phosphorylatable fast, myosin light polypeptide 1, troponin C2 fast, and troponin I skeletal fast 2). Moreover, 7 uncharacterized and 43 novel transcripts differentially expressed by GC and ADX in the hypothalamus have been found. As discussed above, alterations in the expression levels of these identified genes may be important in hypothalamic systems governing energy balance in response to ADX and GC administration. Thus these genes would be potential targets for prevention and/or treatment of anorexia and other forms of wasting illness and obesity caused by deficiency and excess of GC.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Genome Quebec and Genome Canada. M. Yoshioka is supported by the Heart and Stroke Foundation of Canada (HSFC), the Canadian Institute of Health Research (CIHR), and the Canadian Diabetes Association (CDA) as a postdoctoral fellow. J. St-Amand is supported by the Fonds de la recherche en santé du Québec (FRSQ)-Centre Investigator Award.


   ACKNOWLEDGMENTS
 
We thank Drs. Céline Martel and Claude Labrie for procedures with mice, Marc André Rodrigue and Dr. Vincent Raymond for SAGE tag sequencing, Pascal Belleau and Dr. Jean Morissette for bioinformatics, Nathalie Paquet and Dr. Van Luu-The for QRT-PCR experiments, and Dr. Fernand Labrie, the leader of the Atlas of Genomic Profiles of Steroid Action (ATLAS) project.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: J. St-Amand, Functional Genomics Laboratory, CREMO, CHUL, 2705 Blvd Laurier, Quebec, QC, G1V 4G2, Canada (e-mail address: jonny.st-amand{at}crchul.ulaval.ca).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adams MD, Kerlavage AR, Fleischmann RD, Fuldner RA, Bult CJ, Lee NH, Kirkness EF, Weinstock KG, Gocayne JD, White O, et al. Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 377: 3–174, 1995.[Medline]
  2. Amparan D, Avram D, Thomas CG, Lindahl MG, Yang J, Bajaj G, and Ishmael JE. Direct interaction of myosin regulatory light chain with the NMDA receptor. J Neurochem 92: 349–361, 2005.
  3. Aunis D and Bader MF. The cytoskeleton as a barrier to exocytosis in secretory cells. J Exp Biol 139: 253–266, 1988.[Abstract/Free Full Text]
  4. Balkan B, Koylu EO, Kuhar MJ, and Pogun S. The effect of adrenalectomy on cocaine and amphetamine-regulated transcript (CART) expression in the hypothalamic nuclei of the rat. Brain Res 917: 15–20, 2001.[CrossRef][ISI][Medline]
  5. Bambino TH and Hsueh AJ. Direct inhibitory effect of glucocorticoids upon testicular luteinizing hormone receptor and steroidogenesis in vivo and in vitro. Endocrinology 108: 2142–2148, 1981.[Abstract]
  6. Belsham DD and Lovejoy DA. Gonadotropin-releasing hormone: gene evolution, expression, and regulation. Vitam Horm 71: 59–94, 2005.[CrossRef][ISI][Medline]
  7. Berezowsky C and Bag J. Slow troponin C is present in both muscle and nonmuscle cells. Biochem Cell Biol 70: 691–697, 1992.[Medline]
  8. Bridgman PC. Myosin-dependent transport in neurons. J Neurobiol 58: 164–174, 2004.[CrossRef][ISI][Medline]
  9. Brown NS, Smart A, Sharma V, Brinkmeier ML, Greenlee L, Camper SA, Jensen DR, Eckel RH, Krezel W, Chambon P, and Haugen BR. Thyroid hormone resistance and increased metabolic rate in the RXR-{gamma}-deficient mouse. J Clin Invest 106: 73–79, 2000.[ISI][Medline]
  10. Burgess TL and Kelly RB. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol 3: 243–293, 1987.[CrossRef][Medline]
  11. Buss F, Arden SD, Lindsay M, Luzio JP, and Kendrick-Jones J. Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis. EMBO J 20: 3676–3684, 2001.[CrossRef][ISI][Medline]
  12. Carling D. AMP-activated protein kinase: balancing the scales. Biochimie 87: 87–91, 2005.[Medline]
  13. Castellino F, Heuser J, Marchetti S, Bruno B, and Luini A. Glucocorticoid stabilization of actin filaments: a possible mechanism for inhibition of corticotropin release. Proc Natl Acad Sci USA 89: 3775–3779, 1992.[Abstract/Free Full Text]
  14. Castonguay TW, Dallman MF, and Stern JS. Some metabolic and behavioral effects of adrenalectomy on obese Zucker rats. Am J Physiol Regul Integr Comp Physiol 251: R923–R933, 1986.[Abstract/Free Full Text]
  15. Cerignoli F, Guo X, Cardinali B, Rinaldi C, Casaletto J, Frati L, Screpanti I, Gudas LJ, Gulino A, Thiele CJ, and Giannini G. retSDR1, a short-chain retinol dehydrogenase/reductase, is retinoic acid-inducible and frequently deleted in human neuroblastoma cell lines. Cancer Res 62: 1196–1204, 2002.[Abstract/Free Full Text]
  16. Compere V, Li S, Leprince J, Tonon MC, Vaudry H, and Pelletier G. Effect of intracerebroventricular administration of the octadecaneuropeptide on the expression of pro-opiomelanocortin, neuropeptide Y and corticotropin-releasing hormone mRNAs in rat hypothalamus. J Neuroendocrinol 15: 197–203, 2003.[Medline]
  17. Couet J, Martel C, Labrie Y, Luo S, Simard J, and Labrie F. Opposite effects of prolactin and corticosterone on the expression and activity of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in rat skin. J Invest Dermatol 103: 60–64, 1994.[CrossRef][Medline]
  18. Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, Horvath TL, Cone RD, and Low MJ. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411: 480–484, 2001.[CrossRef][Medline]
  19. Dallman MF, la Fleur SE, Pecoraro NC, Gomez F, Houshyar H, and Akana SF. Minireview: glucocorticoids—food intake, abdominal obesity, and wealthy nations in 2004. Endocrinology 145: 2633–2638, 2004.[Abstract/Free Full Text]
  20. Deitmer JW and Rose CR. pH regulation and proton signalling by glial cells. Prog Neurobiol 48: 73–103, 1996.[CrossRef][ISI][Medline]
  21. Del Punta K, Leinders-Zufall T, Rodriguez I, Jukam D, Wysocki CJ, Ogawa S, Zufall F, and Mombaerts P. Deficient pheromone responses in mice lacking a cluster of vomeronasal receptor genes. Nature 419: 70–74, 2002.[CrossRef][Medline]
  22. De Mateos-Verchere JG, Leprince J, Tonon MC, Vaudry H, and Costentin J. The octadecaneuropeptide [diazepam-binding inhibitor (33–50)] exerts potent anorexigenic effects in rodents. Eur J Pharmacol 414: 225–231, 2001.[CrossRef][Medline]
  23. Dinel S, Bolduc C, Belleau P, Boivin A, Yoshioka M, Calvo E, Piedboeuf B, Snyder EE, Labrie F, and St-Amand J. Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome. Nucleic Acids Res 33: e26, 2005.[Abstract/Free Full Text]
  24. Dombrowicz D, Sente B, Closset J, and Hennen G. Dose-dependent effects of human prolactin on the immature hypophysectomized rat testis. Endocrinology 130: 695–700, 1992.[Abstract]
  25. Dong E, Matsumoto K, Tohda M, Kaneko Y, and Watanabe H. Diazepam binding inhibitor (DBI) gene expression in the brains of socially isolated and group-housed mice. Neurosci Res 33: 171–177, 1999.[Medline]
  26. Do-Rego JL, Mensah-Nyagan GA, Beaujean D, Vaudry D, Sieghart W, Luu-The V, Pelletier G, and Vaudry H. {gamma}-Aminobutyric acid, acting through {gamma}-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus. Proc Natl Acad Sci USA 97: 13925–13930, 2000.[Abstract/Free Full Text]
  27. Drazen DL, Coolen LM, Strader AD, Wortman MD, Woods SC, and Seeley RJ. Differential effects of adrenalectomy on melanin-concentrating hormone and orexin A. Endocrinology 145: 3404–3412, 2004.[Abstract/Free Full Text]
  28. Freedman MR, Horwitz BA, and Stern JS. Effect of adrenalectomy and glucocorticoid replacement on development of obesity. Am J Physiol Regul Integr Comp Physiol 250: R595–R607, 1986.[Abstract/Free Full Text]
  29. Garcia-Fernandez LF, Iniguez MA, Eguchi N, Fresno M, Urade Y, and Munoz A. Dexamethasone induces lipocalin-type prostaglandin D synthase gene expression in mouse neuronal cells. J Neurochem 75: 460–470, 2000.[CrossRef][ISI][Medline]
  30. Guidotti A, Forchetti CM, Corda MG, Konkel D, Bennett CD, and Costa E. Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc Natl Acad Sci USA 80: 3531–3535, 1983.[Abstract/Free Full Text]
  31. Guillaume-Gentil C, Assimacopoulos-Jeannet F, and Jeanrenaud B. Involvement of non-esterified fatty acid oxidation in glucocorticoid-induced peripheral insulin resistance in vivo in rats. Diabetologia 36: 899–906, 1993.[CrossRef][ISI][Medline]
  32. Haeseleer F, Huang J, Lebioda L, Saari JC, and Palczewski K. Molecular characterization of a novel short-chain dehydrogenase/reductase that reduces all-trans-retinal. J Biol Chem 273: 21790–21799, 1998.[Abstract/Free Full Text]
  33. Hasson T. Myosin VI: two distinct roles in endocytosis. J Cell Sci 116: 3453–3461, 2003.[Abstract/Free Full Text]
  34. Haugen BR, Jensen DR, Sharma V, Pulawa LK, Hays WR, Krezel W, Chambon P, and Eckel RH. Retinoid X receptor {gamma}-deficient mice have increased skeletal muscle lipoprotein lipase activity and less weight gain when fed a high-fat diet. Endocrinology 145: 3679–3685, 2004.[Abstract/Free Full Text]
  35. Hentges ST, Nishiyama M, Overstreet LS, Stenzel-Poore M, Williams JT, and Low MJ. GABA release from proopiomelanocortin neurons. J Neurosci 24: 1578–1583, 2004.[Abstract/Free Full Text]
  36. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, and Kalra PS. Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 20: 68–100, 1999.[Abstract/Free Full Text]
  37. Kennedy MB. Regulation of neuronal function by calcium. Trends Neurosci 12: 417–420, 1989.[CrossRef][ISI][Medline]
  38. Keverne EB. The vomeronasal organ. Science 286: 716–720, 1999.[Abstract/Free Full Text]
  39. King BM and Smith RL. Hypothalamic obesity after hypophysectomy or adrenalectomy: dependence on corticosterone. Am J Physiol Regul Integr Comp Physiol 249: R522–R526, 1985.[Abstract/Free Full Text]
  40. Kinoshita F, Nakai Y, Katakami H, Imura H, Shimizu T, and Hayaishi O. Suppressive effect of prostaglandin (PG)D2 on pulsatile luteinizing hormone release in conscious castrated rats. Endocrinology 110: 2207–2209, 1982.[Abstract]
  41. Krezel W, Kastner P, and Chambon P. Differential expression of retinoid receptors in the adult mouse central nervous system. Neuroscience 89: 1291–1300, 1999.[CrossRef][ISI][Medline]
  42. Lash AE, Tolstoshev CM, Wagner L, Schuler GD, Strausberg RL, Riggins GJ, and Altschul SF. SAGEmap: a public gene expression resource. Genome Res 10: 1051–1060, 2000.[Abstract/Free Full Text]
  43. Lee K, Li B, Xi X, Suh Y, and Martin RJ. Role of neuronal energy status in the regulation of adenosine 5'-monophosphate-activated protein kinase, orexigenic neuropeptides expression, and feeding behavior. Endocrinology 146: 3–10, 2005.[Abstract/Free Full Text]
  44. Loftus TM, Jaworsky DE, Frehywot GL, Townsend CA, Ronnett GV, Lane MD, and Kuhajda FP. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288: 2379–2381, 2000.[Abstract/Free Full Text]
  45. Luu-The V, Paquet N, Calvo E, and Cumps J. Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction. Biotechniques 38: 287–293, 2005.[ISI][Medline]
  46. Madiehe AM, Lin L, White C, Braymer HD, Bray GA, and York DA. Constitutive activation of STAT-3 and downregulation of SOCS-3 expression induced by adrenalectomy. Am J Physiol Regul Integr Comp Physiol 281: R2048–R2058, 2001.[Abstract/Free Full Text]
  47. Majewska MD, Bisserbe JC, and Eskay RL. Glucocorticoids are modulators of GABAA receptors in brain. Brain Res 339: 178–182, 1985.[CrossRef][ISI][Medline]
  48. Mermall V, Post PL, and Mooseker MS. Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science 279: 527–533, 1998.[Abstract/Free Full Text]
  49. Mong JA, Devidze N, Frail DE, O'Connor LT, Samuel M, Choleris E, Ogawa S, and Pfaff DW. Estradiol differentially regulates lipocalin-type prostaglandin D synthase transcript levels in the rodent brain: evidence from high-density oligonucleotide arrays and in situ hybridization. Proc Natl Acad Sci USA 100: 318–323, 2003.[Abstract/Free Full Text]
  50. Morishita T, Hidaka T, Sugahara K, and Noguchi T. Leptin changes Ca2+/calmodulin-dependent response and up-regulates the gene expression of calcineurin in rat hypothalamus. Life Sci 63: PL311–PL315, 1998.[Medline]
  51. Nishida Y, Yoshioka M, and St-Amand J. The top 10 most abundant transcripts are sufficient to characterize the organs functional specificity: evidences from the cortex, hypothalamus and pituitary gland. Gene 344: 133–141, 2005.[Medline]
  52. Obici S, Feng Z, Arduini A, Conti R, and Rossetti L. Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production. Nat Med 9: 756–761, 2003.[CrossRef][ISI][Medline]
  53. Pantages E and Dulac C. A novel family of candidate pheromone receptors in mammals. Neuron 28: 835–845, 2000.[CrossRef][ISI][Medline]
  54. Paxinos G and Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates (2nd ed.). San Diego, CA: Academic, 2000.
  55. Presse F, Hervieu G, Imaki T, Sawchenko PE, Vale W, and Nahon JL. Rat melanin-concentrating hormone messenger ribonucleic acid expression: marked changes during development and after stress and glucocorticoid stimuli. Endocrinology 131: 1241–1250, 1992.[Abstract]
  56. Rodriguez I, Del Punta K, Rothman A, Ishii T, and Mombaerts P. Multiple new and isolated families within the mouse superfamily of V1r vomeronasal receptors. Nat Neurosci 5: 134–140, 2002.[CrossRef][ISI][Medline]
  57. Romero MF and Boron WF. Electrogenic Na+/HCO3– cotransporters: cloning and physiology. Annu Rev Physiol 61: 699–723, 1999.[CrossRef][ISI][Medline]
  58. Spanswick D, Smith MA, Groppi VE, Logan SD, and Ashford ML. Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature 390: 521–525, 1997.[CrossRef][Medline]
  59. Spanswick D, Smith MA, Mirshamsi S, Routh VH, and Ashford ML. Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats. Nat Neurosci 3: 757–758, 2000.[CrossRef][ISI][Medline]
  60. St-Amand J, Okamura K, Matsumoto K, Shimizu S, and Sogawa Y. Characterization of control and immobilized skeletal muscle: an overview from genetic engineering. FASEB J 15: 684–692, 2001.[Abstract/Free Full Text]
  61. Stricker-Krongrad A and Beck B. Modulation of hypothalamic hypocretin/orexin mRNA expression by glucocorticoids. Biochem Biophys Res Commun 296: 129–133, 2002.[CrossRef][Medline]
  62. Takahata R, Matsumura H, Kantha SS, Kubo E, Kawase K, Sakai T, and Hayaishi O. Intravenous administration of inorganic selenium compounds, inhibitors of prostaglandin D synthase, inhibits sleep in freely moving rats. Brain Res 623: 65–71, 1993.[CrossRef][ISI][Medline]
  63. Tokuyama K and Himms-Hagen J. Enhanced acute response to corticosterone in genetically obese (ob/ob) mice. Am J Physiol Endocrinol Metab 257: E133–E138, 1989.[Abstract/Free Full Text]
  64. Tonon MC, Desy L, Nicolas P, Vaudry H, and Pelletier G. Immunocytochemical localization of the endogenous benzodiazepine ligand octadecaneuropeptide (ODN) in the rat brain. Neuropeptides 15: 17–24, 1990.[CrossRef][Medline]
  65. Trocki O, Baer DJ, and Castonguay TW. Comparison of effects of adrenalectomy and RU-486 in rats given a choice of maintenance diet and fat supplement. Am J Physiol Regul Integr Comp Physiol 269: R708–R719, 1995.[Abstract/Free Full Text]
  66. Urade Y, Fujimoto N, and Hayaishi O. Purification and characterization of rat brain prostaglandin D synthetase. J Biol Chem 260: 12410–12415, 1985.[Abstract/Free Full Text]
  67. Velaz L, Hemric ME, Benson CE, and Chalovich JM. The binding of caldesmon to actin and its effect on the ATPase activity of soluble myosin subfragments in the presence and absence of tropomyosin. J Biol Chem 264: 9602–9610, 1989.[Abstract/Free Full Text]
  68. Velculescu VE, Zhang L, Vogelstein B, and Kinzler KW. Serial analysis of gene expression. Science 270: 484–487, 1995.[Abstract/Free Full Text]
  69. Vrang N, Larsen PJ, Tang-Christensen M, Larsen LK, and Kristensen P. Hypothalamic cocaine-amphetamine regulated transcript (CART) is regulated by glucocorticoids. Brain Res 965: 45–50, 2003.[CrossRef][ISI][Medline]
  70. Wang H, Li S, Givalois L, and Pelletier G. Influence of adrenal glands on the modulation of prolactin gene expression by the endogenous benzodiazepine ligand octadecaneuropeptide in the male rat pituitary gland. J Neuroendocrinol 10: 193–198, 1998.[Medline]
  71. Warrington JA, Nair A, Mahadevappa M, and Tsyganskaya M. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics 2: 143–147, 2000.[Abstract/Free Full Text]
  72. Whitnall MH. Regulation of the hypothalamic corticotropin-releasing hormone neurosecretory system. Prog Neurobiol 40: 573–629, 1993.[ISI][Medline]
  73. Wisse BE, Ogimoto K, Morton GJ, Wilkinson CW, Frayo RS, Cummings DE, and Schwartz MW. Physiological regulation of hypothalamic IL-1ß gene expression by leptin and glucocorticoids: implications for energy homeostasis. Am J Physiol Endocrinol Metab 287: E1107–E1113, 2004.[Abstract/Free Full Text]
  74. Xue J, Douglas RM, Zhou D, Lim JY, Boron WF, and Haddad GG. Expression of Na+/H+ and HCO3–-dependent transporters in Na+/H+ exchanger isoform 1 null mutant mouse brain. Neuroscience 122: 37–46, 2003.[CrossRef][ISI][Medline]
  75. Zakrzewska KE, Cusin I, Sainsbury A, Rohner-Jeanrenaud F, and Jeanrenaud B. Glucocorticoids as counterregulatory hormones of leptin: toward an understanding of leptin resistance. Diabetes 46: 717–719, 1997.[Abstract]
  76. Zakrzewska KE, Cusin I, Stricker-Krongrad A, Boss O, Ricquier D, Jeanrenaud B, and Rohner-Jeanrenaud F. Induction of obesity and hyperleptinemia by central glucocorticoid infusion in the rat. Diabetes 48: 365–370, 1999.[Abstract]
  77. Zhou LW, Moyer JA, Muth EA, Clark B, Palkovits M, and Weiss B. Regional distribution of calmodulin activity in rat brain. J Neurochem 44: 1657–1662, 1985.[CrossRef][Medline]




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