Laura Mikkonen

Päivi Pihlajamaa

Biswajyoti Sahu

Fu-Ping Zhang

Dr Olli A Jänne
email: olli.janne@helsinki.fi

Androgen receptor and androgen-dependent gene expression in lung

Keywords: pneumocyte, gene expression, gene ontology, transcription, glucocorticoid androgen receptor, ARE, androgen response element, ChIP, chromatin immunoprecipitation, Dex, dexamethasone, FBS, fetal bovine serum, GO, gene

The androgen receptor (AR) mediates the effects of male sex steroids. There are major sex differences in lung development and pathologies, including lung cancer. In this report, we show that Ar is mainly expressed in type II pneumocytes and the bronchial epithelium of murine lung and that androgen treatment increases AR protein levels in lung cells. Androgen administration altered significantly murine lung gene expression profiles; for example, by up-regulating transcripts involved in oxygen transport and down-regulating those in DNA repair and DNA recombination. Androgen exposure also affected the gene expression profile in a human lung adenocarcinoma-derived cell line, A549, by up-or downregulating significantly some 200 transcripts, including down-regulation of genes involved in cell respiration. Dexamethasone treatment of A549 cells augmented expression of transcript sets that overlapped in part with those up-regulated by androgen in these cells.

Moreover, a human lung cancer tissue array revealed that different lung cancer types are all AR-positive. Our results indicate that adult lung is an AR target tissue and suggest that AR plays a role in lung cancer biology.

A c c e p t e d M a n u s c r i p t

Introduction

Lung alveoli comprise two epithelial cell types, type I and type II pneumocytes (PTI and PTII, respectively) that enable gaseous exchange. PTI cells form the majority of the epithelium, and while PTII cells account for only about 15% of peripheral lung cells [START_REF] Stone | Allometric relationships of cell numbers and size in the mammalian lung[END_REF], they serve as stem cells that produce new PTI and PTII cells and have other specialized functions. For instance, PTII cells secrete surfactant to reduce the surface tension of the alveoli and decrease the work of breathing by preventing the alveoli from collapsing. Glucocorticoid receptor (GR) is essential in promoting differentiation of PTII cells during embryonic life [START_REF] Ballard | Hormonal regulation of pulmonary surfactant[END_REF][START_REF] Gonzales | Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic[END_REF][START_REF] Gonzales | Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus cAMP[END_REF] and, antenatal glucocorticoid administion expedites lung maturation in infants at risk of preterm delivery [START_REF] Roberts | Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth[END_REF], largely through increased surfactant protein expression [START_REF] Ballard | Hormonal regulation of pulmonary surfactant[END_REF].

Androgen receptor (AR) mediates the effects of male sex steroids in a variety of reproductive and non-reproductive tissues both in males and females under physiological and pathophysiological conditions [START_REF] Dehm | Molecular regulation of androgen action in prostate cancer[END_REF][START_REF] Heemers | Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex[END_REF][START_REF] Heinlein | Androgen receptor (AR) coregulators: an overview[END_REF][START_REF] Heinlein | Androgen receptor in prostate cancer[END_REF]. Lungs of male fetuses develop more slowly than those of females, and males are more prone to neonatal respiratory distress syndrome due to reduced number of PTII cells and lack of surfactant [START_REF] Perelman | Discordance between male and female deaths due to the respiratory distress syndrome[END_REF]. The AR has been studied in the developing human and murine lungs, and it has been shown to influence branching morphogenesis [START_REF] Kimura | Expression of androgen receptor and 5-reductase types 1 and 2 in early gestation fetal lung: a possible correlation with branching morphogenesis[END_REF] and to attenuate PTII cell maturation [START_REF] Dammann | Androgen regulation of signaling pathways in late fetal mouse lung development[END_REF][START_REF] Provost | Androgen formation and metabolism in the pulmonary epithelial cell line A549: expression of 17-hydroxysteroid dehydrogenase type 5 and 3hydroxysteroid dehydrogenase type 3[END_REF]. During development, there are several differentially expressed genes in the lungs of female and male mouse fetuses, including apolipoproteins that may be involved in local lipid metabolism and transport related to surfactant lipid synthesis [START_REF] Simard | Sexually dimorphic gene expression that overlaps maturation of type II pneumonocytes in fetal mouse lungs[END_REF]. However, the effect of androgens on the gene expression profile in adult lung has not been assessed.

Lung cancer is a disease with a clear sex difference. Female patients show better survival rates than males at any stage of the disease [START_REF] Fu | Lung cancer in women: analysis of the national surveillance, epidemiology, and end results database[END_REF]. Histological subtypes of the disease in women include proportionally more adenocarcinoma and less squamous cell carcinoma than men [START_REF] Chen | Distribution according to histologic type and outcome by gender and age group in Taiwanese patients with lung carcinoma[END_REF][START_REF] Fu | Lung cancer in women: analysis of the national surveillance, epidemiology, and end results database[END_REF]. Overall, men appear to have a higher rate of fatal outcome of lung cancer, but tend to be less vulnerable to tobacco carcinogens than women (International Early Lung Cancer Action Program Investigators et al., 2006). It remains to be elucidated whether these differences result from men A c c e p t e d M a n u s c r i p t 4 having lower circulating female sex steroid (estrogen and progesterone) or higher androgen levels than women, or from some as yet unknown confounding associations.

There is a previous report on the presence of AR in adult human lung [START_REF] Wilson | A and B forms of the androgen receptor are expressed in a variety of human tissues[END_REF], but very little is known about functional importance of AR and androgen signaling in lung physiology after embryonal development. To address these issues, we have examined expression of the Ar gene and searched for androgen-dependent target genes in adult murine lung. Androgen-treated A549 cells, representing a transformed human PTII adenocarcinoma cell line, were used to compare androgen-dependent gene expression profiles in human cells with those in murine lung. Since androgens and glucocorticoids are known to regulate expression of the same genes, such as the prostate-specific antigen gene (Cleutjens et al., 1977;[START_REF] Thompson | Small carboxyl-terminal domain phosphatase 2 attenuates androgendependent transcription[END_REF], the A549 cells were also employed to examine similarities and differences between gene expression profiles regulated by androgens and glucocorticoids through the AR and GR, respectively.

Materials and methods

Mice and hormone treatments

Wild-type FVB mice were used. One week after castration, one group of male mice received androgen for five days [1 mg testosterone (T)/mouse/day in 0.1 ml of mineral oil as sc injections, corresponding to 30 µg T/g of body weight], whereas the control group received only vehicle. The dose of the androgen was based on similar previous experiments with mice [START_REF] Pajunen | Androgenic regulation of ornithine decarboxylase activity in mouse kidney and its relationship to changes in cytosol and nuclear androgen receptor concentrations[END_REF]. Animals were sacrificed on day six, and the lungs were collected for RNA and protein isolation as well as for immunohistochemical analysis. The same hormone treatment (30 µg T/g of body weigth for 5 days) was given in some experiments to intact females. All results originate from a minimum of three biological replicate experiments. All the experiments were performed according to the guidelines for animal experiments at the University of Helsinki and under the license from appropriate Finnish Review Boards for Animal Experiments. For hormone exposure experiments, cells were cultured in charcoal-stripped 10% FBS for 24 h and then treated either with 100 nM T or 1 µM dexamethasone (Dex) in charcoalstripped 2% FBS for 24 h. The hormone concentrations used were similar to those used by other investigators (So et al., 2007b;[START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF]. All results are representative of at least two independent experiments.

Cells

Protein isolation and immunoblotting

Proteins were isolated from cells or homogenized tissues using a buffer containing 50 mM Tris-HCl (pH 7.8), 300 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM Nethylmaleimide (Sigma-Aldrich, St. Louis, MO), and 1 x complete protease inhibitor set (Roche Diagnostics, Indianapolis, IN). Lysates were centrifuged, and soluble protein concentration was quantified with the BioRad protein assay. Protein samples (50 g or 30 g) were resolved on a 10% SDS-PAGE gel and transferred onto ECL membrane (GE Healthcare, Buckinghamshire, England). Immunoblotting was performed with rabbit polyclonal IgG against AR (sc-816, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit polyclonal IgG against GR (sc-1004, Santa Cruz Biotechnology) or mouse monoclonal tubulin antibody (sc-5286, Santa Cruz Biotechnology). Horseradish peroxidaseconjugated goat anti-mouse or anti-rabbit IgG was used as secondary antibody. ECL reagent (GE Healthcare) was used for visualization. Band intensities were quantified using the Kodak 1D Scientific Imaging Systems version 3.5.3.

Immunohistochemistry

Lung and prostate tissue samples were fixed immediately after dissection in 4% paraformaldehyde at 4°C overnight, dehydrated and embedded in paraffin. Five-µm sections were mounted onto Superfrost Plus slides (Menzel GmbH, Braunschweig, Germany), dewaxed with xylene, rehydrated and the endogenous peroxidase activity blocked by incubation in 3% hydrogen peroxide for 15 min. Antigen retrieval was carried out by boil-A c c e p t e d M a n u s c r i p t 6 ing the slides in 10 mM sodium citrate (pH 6.0). The slides were subsequently blocked with 1% bovine serum albumin (Sigma-Aldrich) and normal goat serum (Vector Laboratories, Burlingame, CA), and incubated with antibodies against AR (1:1 000 dilution, sc-816, Santa Cruz Biotechnology), GR (1:2 000 dilution, sc-1004, Santa Cruz Biotechnology) or hemoglobin // (1:100 dilution, sc-21006, Santa Cruz Biotechnology) overnight at 4°C. The slides were then incubated with biotinylated anti-rabbit IgG secondary antibody as a 1:200 dilution (Vector Laboratories). Negative controls with normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and in the absence of the primary antibody were included in each experiment. The immune complexes were visualized using the Vectastain Elite ABC and DAB substrate kits (Vector Laboratories). A set of slides were counterstained using Mayer's hemalum solution (Merck, Darmstadt, Germany). The slides were dehydrated and mounted using Permount (Fisher Chemicals, Fair Lawn, NJ).

Tissue Array

A206 (Lung Cancer Tissues) AccuMax Array containing total 84 spots including different types of lung carcinoma was purchased from Petagen (Seoul, Korea). The array was immunostained with anti-AR-antibody (sc-816, Santa Cruz Biotechnology) as described above. Another polyclonal AR antibody raised against full-length human receptor (AR3, [START_REF] Thompson | Small carboxyl-terminal domain phosphatase 2 attenuates androgendependent transcription[END_REF] was used to confirm the results.

RNA isolation and blotting

Total RNA was isolated from cells or homogenized tissues using TRIzol reagent (Invitrogen) or Nucleospin RNA II kit (Macherey-Nagel GmbH, Düren, Germany) according to the manufacturers' instructions. Ten µg of total RNA from different mouse tissues were resolved on a 1.2% denaturing agarose gel and transferred onto a Hybond XL nylon membrane (GE Healthcare). The blot was hybridized with a 32 P-labeled anti-sense RNA probe corresponding to nucleotides 1301-1655 of Ar cDNA (NM_013476), generated by using Riboprobe Synthesis II kit (Promega, Madison, WI). Hybridization was carried out at 68°C in Ultrahyb buffer (Ambion, Austin, TX) overnight. The membrane was washed in 2 x SSC, 0.1% SDS and 0.1 x SSC, 0.1% SDS and exposed to Fuji X-ray film at -70°C.

A c c e p t e d M a n u s c r i p t 7

Microarray analysis

The quality of RNA was assessed by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) prior to being subjected to microarray experiments, and the RNA integrity number was > 8 for each sample. Five g of total RNA samples from lungs of T-treated castrated male mice (n=3), vehicle-treated castrated male mice (n=3), intact male mice (n=3), intact female mice (n=3), non-treated A549 cells (n=2), T-treated A549 cells (n=2) or Dex-treated A549 cells (n=2) were amplified and labeled to produce cRNA using the Low RNA Input Linear Amp Kit PLUS, Two-Color (Agilent). cRNA was hybridized onto G4122F Whole Mouse Genome chips or G4112F Whole Human Genome chips (Agilent) comprising over 40 000 mouse or human genes and transcripts, respectively, according to the manufacturer's instructions. The chips were scanned with Agilent DNA microarray scanner and Feature Extraction Software using Linear and Lowess normalizations. Each chip compared the relative abundance of the transcripts (i.e., abundance differences in the sample pairs) in either T-and vehicle-treated lungs (G4122F chips), intact male and female lungs (G4122F chips), T-and vehicle-treated A549 cells (G4112F chips) or Dexand vehicle-treated A549 cells (G4112F chips).

Microarray results were analyzed with Genespring GX 7.3.1 (Agilent). Data were normalized per spot and per chip as recommended by the manufacturer, and spots flagged as absent in more than half of the samples were removed. Transcripts with fold-change >1.8 and Student's t-test p-value <0.05 were considered significantly differentially expressed. Genes that were reported by several spots have been kept separate and may appear more than once in the lists. Moreover, the transcripts that did not have a current annotation were removed from the final lists. The resulting lists were analyzed further with Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt) by organizing the lists into Gene Ontology (GO) categories and comparing the categories with those expected by random selection from the whole genome. Original microarray data sets are available at www.ebi.ac.uk with accession numbers E- MEXP-2001, E-MEXP-2002, E-MEXP-2003, and E-MEXP-2004.

Quantitative reverse transcription PCR (qRT-PCR)

A c c e p t e d M a n u s c r i p t 8 cDNA was synthesized from 3 g of total lung RNA samples using random hexamer primers and Superscript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions. qRT-PCR was performed with LightCycler® 480 Real-Time PCR System (Roche Diagnostics) in 20-l reactions containing LightCycler® 480 SYBR Green I Master (Roche Diagnostics) and 1 M forward and reverse primers (see Supplemental data, Table S1). PCR reactions included a 5-min denaturation step 95°C followed by 40 cycles of 10-s denaturation at 95°C, 5-s annealing at 57-60°C, 20-s extension at 72°C and 5-s SYBR Green signal measurement. Melting curve analysis was performed for each product to detect possible non-specific products. The results were analyzed with LightCycler analysis software (Roche Diagnostics). The results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed essentially as previously described [START_REF] Kang | Coregulator recruitment and histone modifications in transcriptional regulation by the androgen receptor[END_REF][START_REF] Thompson | Small carboxyl-terminal domain phosphatase 2 attenuates androgendependent transcription[END_REF]. Briefly, 10 6 A549 cells were seeded on 10-cm dishes and maintained for 4 days in 10% charcoal-stripped FBS, F-12K Nutrient Mixture medium. The cells were exposed to either 100 nM T or vehicle for 16 h and then cross-linked in 1% formaldehyde. The cells were harvested into lysis buffer and pelleted by centrifugation. Chromatin was sonicated to an average DNA length of 500-800 nt. Sonicated samples were precleared with normal rabbit IgG and GammaBind G Sepharose (GE Healthcare) and immunoprecipitated with AR antiserum (sc-816, Santa Cruz Biotechnology). Antibody-bound complexes were adsorbed to GammaBind G Sepharose beads that were sequentially washed with TSE I, TSE II, TSE III, and TE buffers [START_REF] Kang | Coregulator recruitment and histone modifications in transcriptional regulation by the androgen receptor[END_REF]. DNA was eluted from the matrix with 1% SDS in 0.1 M NaHCO 3 , crosslinking was reverted at 65C overnight, and DNA isolated using QIAquick PCR purification system (Qiagen GmbH, Hilden, Germany). Input samples were treated the same way except that no immunoprecipitation was performed. qRT-PCR was performed with LightCycler 480 System in 20-l reactions containing LightCycler 480 SYBR Green I Master and 1 M forward and reverse primers for the genomic regions examined (Supplemental data, Table S1). PCR reactions included a 5-min denaturation step at 95°C, followed by 40 cycles of 10-s denaturation at 95°C, 5-s annealing at 60°C, 5-s extension at

Results

Lung cell expression of the Ar gene

Ar mRNA was detected in hybridization blots of total murine lung RNA as a distinct ~10-kb band. The presence of Ar mRNA size heterogeneity has been reported previously [START_REF] Tilley | Expression of the human androgen receptor gene utilizes a common promoter in diverse human tissues and cell line[END_REF][START_REF] Shan | Regulation of androgen receptor protein and messenger RNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells[END_REF], with two mRNA species of ~10 kb and ~8 kb in size. This latter Ar mRNA species was barely detectable in murine lung but corresponded to the principal Ar mRNA from in brain (Fig. 1A). The abundance of Ar mRNA in lung was comparable to that in brain, but less than in kidney or prostate. This result was confirmed with RT-PCR (data not shown). Five-day T treatment increased AR protein levels in lungs of both castrated males and intact females, as revealed by immunoblotting of tissue extracts (Fig. 1B), and the amount of AR in the castrated male lung appeared to be higher than that in female lung. Likewise, lung AR protein level was lower in castrated than in intact male mice (Figs. 1C,1D). Up-regulation of AR levels either by physiological or pharmacological amounts of androgen is likely to be due to ligand-dependent stabilization of AR, as has been found in other AR-containing tissues or cells [START_REF] Shan | Regulation of androgen receptor protein and messenger RNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells[END_REF][START_REF] Kemppainen | Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones[END_REF][START_REF] Zhou | Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability[END_REF][START_REF] Mora | Autoregulation of androgen receptor protein and messenger RNA in rat ventral prostate is protein synthesis dependent[END_REF]. In support to this notion, five-day T treatment failed to alter significantly Ar mRNA levels in murine lung (results not shown). As reported previously [START_REF] Wollmer | Steroid-hormone receptors in cell lines and tumor biopsies of human lung cancer[END_REF], we detected AR protein in a human cancer cell line derived from PTII cells (the A549 cell line), and upon androgen exposure, A549 cell AR protein level was increased in a fashion similar to that in murine lung (Fig. 1E).

AR antigen was detected by immunohistochemistry in nuclei of murine PTII and bronchial epithelial cells of intact male mice (Fig. 2A). In these experiments, a murine prostate sample served as a positive control, and showed clear staining of the epithelial cell nuclei (Supplemental Fig. S1). In comparison, GR immunoreactivity exhibited a more universal distribution than that of AR in the same tissue sections, and all lung cell types expressed GR antigen (Fig. 2B). Upon androgen treatment of castrated male mice, AR immunostaining of the lung became more intense, but the subcellular localization of AR A c c e p t e d M a n u s c r i p t 10 antigen did not change significantly (Figs. 2C and2D), whereas the intensity of GR immunostaining was independent of androgen exposure (data not shown).

Androgen treatment affects gene expression profile in murine lung

In search of androgen-regulated genes in murine lung, we performed gene expression profiling using the Agilent Whole Mouse Genome platform. By using the cut-off values of fold-change >1.8 and p value <0.05, we identified 46 up-regulated and 134 downregulated transcripts in lung of castrated male mice after T treatment (Supplemental data, Table S2). To infer the biological processes potentially regulated by androgens, the genes were grouped into gene ontology (GO) categories. These categories were compared with the expected ones by using the Webgestalt mouse as a reference gene set, and the hypergeometric test was used to calculate the p values. Among the enriched categories in the up-regulated genes were those involved in oxygen transport (4 genes, p=3.59e-8), negative regulation of apoptosis (3 genes, p=3.79e-3), hemopoiesis (3 genes, p=8.02e-3) and heme biosynthesis (2 genes, p=2.14e-4). The down-regulated genes belonged to categories such as DNA repair (5 genes, p=6.65e-3), DNA recombination (3 genes, p=9.19e-3), and double-strand break repair (2 genes p=4.20e-3) (Table 1). Quantitative RT-PCR was used to confirm expression levels of a few selected transcripts, and all transcript levels quantified by qPCR were in good agreement with the microarray results (Table 2).

To examine sexual dimorphism in lung gene expression and to search for genes potentially regulated by physiological androgen concentrations, gene expression profiling was performed on male and female lungs. With the same cut-off values as above (foldchange >1.8 and p value <0.05), we identified 28 up-regulated and 99 down-regulated transcripts in male lungs as compared to female lungs (Supplemental data, Table S3). Some of the up-regulated genes in males belonged to muscle development (3 genes, p=2.28e-4) and muscle contraction (6 genes, p=3.78e-11). The down-regulated genes included genes involved in acute inflammatory response (3 genes, p=6.05e-3) and regulation of translation (2 genes, p=4.58e-2) (Table 3). As could have been expected, Y chromosome-encoded transcripts were highly overrepresented in the male samples. These included genes such as Eif2s3y, Ddx3y and Uty. Xist, the X inactive specific transcript in females, was at the top of the list of down-regulated genes in males. The gene lists covering the two data sets -T-treated lungs vs. non-treated lungs of castrated male mice, and intact male vs. intact female lungsexhibited very limited overlap. There were 7 overlapping down-regulated transcripts that included 6 genes: Kcnj8, Txk, D0H4S114, Hsd17b12, BC002199, and Tmem46. The up-regulated lists did not overlap when the cut-off was set at fold change of 1.8. However, when the cut-off value was relaxed to fold change of 1.5, the lists of up-regulated genes overlapped by 9 transcripts (7 genes: Myl7, Hrc, Tnnt2, Mb, Fgl1, Cdh2 and Junb).

Gene expression profiling experiments revealed androgen-dependent up-regulation of several hemoglobin-related transcripts (Hbb-b1, Hbb-y, Hbq1, Mb) in the murine lung.

To examine whether up-regulation was due to increased expression of these genes by lung cells or increased blood cell retention in lung as a consequence of T administration, we performed immunohistochemical analyses with an antibody recognizing several hemoglobin variants (,  and ) on murine lung tissue sections. This antibody stained primarily the nuclei of all lung cell types, and T treatment appeared to bring about an increase in signal intensity within these cells (Fig. 2E andF).

AR is present in normal and malignant human lung cells

Lung carcinoma tumors and normal adjacent tissue showed AR immunoreactivity in normal lung, and all lung cancer types within the array included at least a few samples that were clearly AR-positive (Fig. 3). AR staining was mainly nuclear, although diffuse cytoplasmic staining was also present in many samples. The tissue array comprised samples from adenocarcinoma (7 AR-positive of 15 samples), squamous cell carcinoma (11/15), bronchioloalveolar carcinoma (2/3), large cell neuroendocrine carcinoma (2/2), large cell carcinoma (2/3), and small cell carcinoma (2/2). The staining of the tumor was not dependent on the sex of the patient. All normal lung tissue specimens in the array exhibited clear AR immunoreactivity, but the signal intensity varied among the samples. An other AR antibody [START_REF] Thompson | Small carboxyl-terminal domain phosphatase 2 attenuates androgendependent transcription[END_REF] was used to confirm the immunostaining results. S4). Of the known AR target genes, the list included TMPRSS2 and EPB41L4B [START_REF] Bolton | Celland gene-specific regulation of primary target genes by the androgen receptor[END_REF][START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF].

Androgen exposure affects gene expression of A549 cells

The genes belonged to the following GO categories: phospholipid metabolism (2 genes, p=0.03), iron ion binding (3 genes, p=0.046), oxygen binding (2 genes, p=0.0057), heme binding (3 genes, p=0.0028) and clathrin-coated vesicle (2 genes, p=0.03) (Supplemental data, Table S5). T elicited down-regulation of 111 transcripts, 98 of which corresponded to currently known genes (Supplemental data, Table S4), and these included the following GO categories: cellular respiration (3 genes, p=0.0006), metabolism (44 genes, p=0.009), mitochondrion (8 genes, p=0.008) and clathrin coat of trans-Golgi network vesicle (3 genes, p=0.00004). Among the enriched categories of down-regulated genes was also regulation of apoptosis (6 genes, p=0.008); these genes included both negative regulation (3 genes, p=0.02) and positive regulation of apoptosis (4 genes, p=0.009) (Supplemental data, Table S5). Unlike mouse lung in vivo, expression of hemoglobin genes was not significant up-regulated by androgen in A549 cells. This result may relate to the fact that expression levels of all hemoglobin genes were extremely low in A549 cells.

AR regulates gene expression through canonical androgen response elements (AREs) and non-canonical AREs [START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF], and class I nuclear receptors, such as AR, GR, progesterone receptor, and mineralocorticoid receptor, may recognize the same canonical response elements (for a review, see [START_REF] Verrijdt | Selective DNA recognition by the androgen receptor as a mechanism for hormone-specific regulation of gene expression[END_REF]. It was, therefore, of interest to expose A549 cells to Dex and compare AR and GR target genes in the context of a single cell line. As expected, more A549 transcripts were regulated by Dex than T exposure. Likewise, Dex responses were more robust than those with T. With the cut-off value of 2-fold change, Dex up-regulated the abundance of 1510 transcripts (compared to 74 with T at the same fold change), and 1285 transcripts were down-regulated by Dex (compared to 56 with T). Nevertheless, there was some overlap in transcript regulation, in that 40 transcripts were common for T and Dex among the up-regulated genes, whereas only 11 common ones were among the down-regulated genes (Fig. 4). Among the most Dex-responsive genes [>3-fold up-regulation (510 transcripts corresponding to 416 known genes); Supplemental data, Table S6] were those involved in lipid metabolism (23 genes, p=9.8e-5), suggesting a possible increase in surfactant production (Supplemental S7). A few of the surfactant-related genes were up-regulated in Dex-treated cells, such as SFTPD (10.4-fold), SFTPG (3.7-fold), and PGC (12.2-fold). Some other genes encoding surfactant-associated proteins were expressed at very low abundance and were, therefore, flagged in the analysis as being absent. Genes down-regulated by Dex (225 transcripts, 209 known genes) included those in stress response (15 genes, p=7e-3), DNA metabolism (19 genes, p=5e-5), and mitosis (12 genes, p=8e-8) (Supplemental data, Table S7).

The transcripts that were up-regulated at least 2-fold by both T and Dex included several interesting genes that are listed in Table 4. One of the genes listed in this table, TMPRSS2, is known to harbor an androgen-responsive enhancer at ~14 kb of transcription start site [START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF]. The TMPRSS2 locus plays also an important role in prostate cancer biology, as it is a common place for translocation of genes encoding members of the ETS transcription factor family [START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF]. In view of this, it was of interest to observe that, in A549 cells, there was androgen-dependent loading of AR onto the TMPRSS2 enhancer, as revealed by ChIP assays (Fig. 5). There is no other information on potential AR-binding sites in the regulatory regions of androgen-regulated genes in A549 cells. However, our ongoing experiments on LNCaP-1F5 prostate cancer cell line have employed ChIP with AR antibody followed by ultrahigh-throughput parallel sequencing (ChIP-seq) to delineate genome-wide AR-binding sites on chromatin (our unpublished observations). Even though this AR-binding site information originates from a different cell line, it was still pertinent to examine their occurrence in androgen-regulated genes of the A549 cells. We found that 39% of androgen up-regulated and 36% of down-regulated genes contained putative AR-dinding sites within 200 kb of transcription start sites. On the basis of this analysis, additional ChIP assays were performed on two other loci, an ARbinding site at 21 kb upstream of the TAS2R50 gene encoding a G-protein coupled taste receptor and a negative control site at 57 kb down-stream of the PDGFRA gene. Upon androgen exposure for 16 h, there was significantly increased loading of AR on the former but not onto the latter site (Fig. 5). Up-regulation of TMPRSS2 and TAS2R50 mRNA levels upon T treatment was confirmed by qPCR (Supplemental data, Table S3).

Localization of AR expression to specific lung cell types

In addition to fetal lung [START_REF] Dammann | Androgen regulation of signaling pathways in late fetal mouse lung development[END_REF][START_REF] Kimura | Expression of androgen receptor and 5-reductase types 1 and 2 in early gestation fetal lung: a possible correlation with branching morphogenesis[END_REF][START_REF] Provost | A link between lung androgen metabolism and the emergence of mature epithelial type II cells[END_REF], we have now shown that adult lung is an androgen-responsive tissue in both mice and humans, and the AR is expressed predominantly in the bronchial epithelium and PTII cells. The latter cells are important in the maintenance of alveolar epithelium by (i) providing new epithelial cells and (ii) secreting surfactant. Since T delays maturation of PTII cells during embryonic development [START_REF] Provost | A link between lung androgen metabolism and the emergence of mature epithelial type II cells[END_REF], it is not surprising that Ar is also expressed in these cells in adult mice. In view of the specific role of PTII cells in surfactant production, AR might also influence surfactant biosynthesis, but there are no reports to indicate that androgen insensitive Tfm/Y mice or human androgen insensitive patients have a lung phenotype. Moreover, although lung surfactant composition of Tfm/Y mice differs from that of wild-type mice during development [START_REF] Nielsen | Androgen receptors influence the production of pulmonary surfactant in the testicular feminization mouse fetus[END_REF], we failed to observe significant androgen-dependent differences in transcripts encoding surfactant proteins in adult lung, between either T-treated and castrated male mice or intact male and female mice.

Androgen-regulated gene expression in mouse lung and A549 cells

Androgen-dependent genes in the murine lung include those in iron binding and oxygen transport and, in particular, many hemoglobin-related gene transcripts. Hemoglobin genes are expressed, not only in blood cells, but also in cultured PTII and Clara cells [START_REF] Newton | Hemoglobin is expressed by alveolar epithelial cells[END_REF], and cellular hemoglobin is proposed to serve as a protective agent against oxidative and nitrosative stress [START_REF] Liu | Protection from nitrosative stress by yeast flavohemoglobin[END_REF][START_REF] Minning | Ascaris haemoglobin is a nitric oxide-activated 'deoxygenase[END_REF]. Androgen treatment not only increased expression of genes involved in oxygen transport, such as those encoding hemoglobins, but it also decreased expression of genes involved in DNA damage repair. It is tempting to speculate that cells with increased protection against oxidative stress would require less DNA repair proteins. Case-control studies have shown that women have an increased risk of lung cancer over that in men with the same consumption of tobacco [START_REF] Risch | Are female smokers at higher risk for lung cancer than male smokers? A case-control analysis by histologic type[END_REF][START_REF] Zang | Differences in lung cancer risk between men and women: examination of the evidence[END_REF]. Interestingly, one of the genes that was most down-regulated in male vs. female murine lung was Cyp1a1 that encodes an A c c e p t e d M a n u s c r i p t enzyme involved in metabolism of polycyclic aromatic hydrocarbons. Increased expression of this enzyme in women has been proposed to play a role in their elevated lung cancer risk [START_REF] Mollerup | Sex differences in lung CYP1A1 expression and DNA adduct levels among lung cancer patients[END_REF].

Several androgen-regulated genes did not respond in lung in the same ways as in some other androgen target tissues. Tissue-specific differences are not surprising, since gene expression depends on chromatin modifications, coactivators and corepressors typical of a given cellular environment. For example, Igf1 was significantly down-regulated in lung, whereas it is up-regulated in other androgen target tissues [START_REF] Bolton | Celland gene-specific regulation of primary target genes by the androgen receptor[END_REF]. In addition, Angptl4, a gene that is linked to lung cancer metastasis seeding via the TGF- signaling pathway [START_REF] Padua | TGF primes breast tumors for lung metastasis seeding through angiopoietin-like 4[END_REF], is down-regulated by androgen in prostate cancer cells [START_REF] Bolton | Celland gene-specific regulation of primary target genes by the androgen receptor[END_REF] but up-regulated in murine lung. Up-regulation of the ANGPTL4 gene has also been linked to hypoxic conditions in human fetal cardiac cells and neonatal rat cardiomyocytes [START_REF] Belanger | Hypoxia up-regulates expression of peroxisome proliferatoractivated receptor gamma angiopoietin-related gene (pgar) in cardiomyocytes: role of hypoxia inducible factor 1[END_REF]. This, together with up-regulated hemoglobin genes, could relate to the ways by which the lung reacts to changing oxygen levels.

Another interesting gene that was one of the most androgen down-regulated genes in murine lung is D0H4S114, also known as P311, which has a role in distal lung morphogenesis during development, and its absence in adults may result in emphysema [START_REF] Zhao | Identification of P311 as a potential gene regulating alveolar generation[END_REF].

Many genes that were androgen-regulated in A549 cells are known AR targets in other tissues. These genes include TMPRSS2 (up-regulated by androgens in prostate cancer, [START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF][START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF] and EPB41L4B [START_REF] Bolton | Celland gene-specific regulation of primary target genes by the androgen receptor[END_REF]. Similar to mouse lung, the list of up-regulated genes in A549 cells included significant enrichment of GO categories dealing with oxygen binding, heme binding and iron ion binding, even though the genes whose expression was modulated by androgens were not the same. Collectively, our results imply that androgens play a role in transport and utilization of oxygen in lung.

Although the enriched GO categories showed similarity between murine lung and human A549 lung cancer cells, differentially expressed single genes were not the same.

However, A549 cells represent a malignant human cell line and may not reflect all functions of intact PTII cells. In addition, PTII cells are not the only lung cells expressing AR.

As the lung tissue represents the first-line defense between the body and its surroundings, environmental conditions ought to play a more significant role in modulating gene expres-

Comparison of T and Dex regulation of A549 cell gene expression profiles

Glucocorticoid receptor (Gr) null mice die during the first few hours after birth due to respiratory failure [START_REF] Cole | Targeted disruption of the gluco-corticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation[END_REF], and GR protein is expressed in all cell types of the lung. To examine a potential overlap between androgen-and glucocorticoiddependent genes in lung cells, we compared gene expression profiles of A549 cells exposed to either T or Dex. Most of the genes identified previously by chromatin immunoprecipitation-microarray (ChIP-on-chip) studies in Dex-treated A549 cells (So et al., 2007a) were included in our list of differentially expressed (>2-fold) genes in Dex-treated cells. The majority of genes regulated by T or Dex in A549 cells were distinct, indicating that AR possesses functions that are independent of GR signaling in lung cells. Nevertheless, gene expression profiles from T-and Dex-treated A549 cells also showed the presence of overlapping sets of transcripts, and it is therefore likely that some of the androgenresponsive genes are also regulated by GR signaling. Interestingly, the majority of these genes were up-regulated by both T and Dex. Whether or not this is due to binding of ligand-occupied AR and GR to the same genomic elements remains to be elucidated.

AR and lung cancer

There are several reports implicating the estrogen signaling pathway in lung cancer [START_REF] Marquez-Garban | Estrogen receptor signaling pathways in human non-small cell lung cancer[END_REF], and clear sex differences exist in lung cancer type and survival [START_REF] Belani | Women and lung cancer: epidemiology, tumor biology, and emerging trends in clinical research[END_REF][START_REF] Chen | Distribution according to histologic type and outcome by gender and age group in Taiwanese patients with lung carcinoma[END_REF][START_REF] Fu | Lung cancer in women: analysis of the national surveillance, epidemiology, and end results database[END_REF][START_REF] Patel | Lung cancer in women[END_REF][START_REF] Thomas | Lung cancer in women: emerging differences in epidemiology, biology, and therapy[END_REF]. In particular, women who do not smoke are more susceptible to developing adenocarcinoma, a cancer type that derives from PTII cells expressing AR. Since epidermal growth factor receptor (EGFR) plays a major role in adenocarcinoma [START_REF] Johnson | Epidermal growth factor receptor mutations in patients with non-small cell lung cancer[END_REF], it is interesting that an inhibitor of EGFR which is widely used to treat lung adenocarcinoma, gefitinib, has been shown to lower androgen levels especially in patients responding to treatment [START_REF] Nishio | Gefitinib treatment affects androgen levels in non-smallcell lung cancer patients[END_REF]. Our results show that some human lung cancers express AR, and a human alveolar carcinoma-derived cell line with PTII-like properties, A549, not only contains AR but also exhibits androgen-dependent gene expression. Some A c c e p t e d M a n u s c r i p t of the androgen-responsive genes in these cells belong to interesting categories with regard to survival of malignant cells, such as oxygen utilization and apoptosis. This result combined to androgen-responsive genes in murine lung, involving iron ion binding, DNA repair and angiogenesis, makes the role of androgens in lung cancer biology worthy of further inquiry. In this regard, TMPRSS2 was one of the genes that was up-regulated upon androgen exposure and a direct AR target in A549 cells, as judged by ChIP experiments.

This gene is also an AR target in prostate [START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF], and a locus for translocation of ETS transcription factors in about 50% of prostate cancers [START_REF] Kumar-Sinha | Recurrent gene fusions in prostate cancer[END_REF][START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF]. It is currently not known whether a similar translocation takes place in a subset of lung cancers. With regard to lung cancer development and metastasis seeding, it was of particular interest to find androgen-dependent up-regulation of Angptl4 in murine lung, as this TGF- regulated gene has been implicated in lung cancer progression and metastatic potential [START_REF] Padua | TGF primes breast tumors for lung metastasis seeding through angiopoietin-like 4[END_REF]. This gene is also a PPAR target [START_REF] Yoon | Peroxisome proliferator-activated receptor gamma target gene encoding a novel angiopoietin-related protein associated with adipose differentiation[END_REF], and PPAR agonists reduce the growth of non-small cell lung cancer derived cellsincluding adenocarcinoma [START_REF] Chang | Induction of differentiation and apoptosis by ligands of peroxisome proliferator-activated receptor gamma in non-small cell lung cancer[END_REF] in vitro, and patients with diabetes using PPAR agonists have a reduced risk of lung cancer [START_REF] Govindarajan | Thiazolidinediones and the risk of lung, prostate, and colon cancer in patients with diabetes[END_REF]. In conclusion, our data suggest that AR, along with other nuclear receptors, plays a role in lung cancer biology. RNA from different mouse tissues were resolved on a 1.2% formaldehyde denaturing agarose gel, transferred onto a nylon membrane and hybridized with a 32 P-labeled Ar cRNA probe as described in the Materials and Methods. B, Immunoblot analysis of AR protein in murine lung. Female or castrated male mice were treated testosterone (T) for 5 days; control animals received vehicle only. Soluble lung proteins (50 µg) were resolved by SDS-PAGE gel electrophoresis and immunoblotted using antibodies against AR or GR. C, AR protein levels in the lung of intact and castrated male mice. Soluble protein extracts (50 µg protein) were resolved by SDS-PAGE gel electrophoresis and immunoblotted using antibodies against AR D, A549 cells were treated with 100 nM testosterone (T) for 24 h. Total protein (50 µg) extracts were resolved on an SDS-PAGE and immunoblotted with an antibody against AR. -Tubulin was used to control for equal loadings. Table 1. Enriched gene ontology (GO) categories in murine lung upon T treatment Agilent Whole Mouse Genome expression microarray was used to compare gene-expression in lungs of castrated male mice treated with testosterone for five days to those of vehicle-treated animals. The GO categories are shown for transcripts that were differentially expressed (fold-change >1.8, t-test p value <0.05).

Up-regulated

  derived A549 cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in F-12K Nutrient Mixture, Kaighn's Modification (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS). This cell line originates from explant culture of lung carcinomatous tissue from a Caucasian male.

  s SYBR Green signal measurement. The immunoprecipitated samples were normalized to input values.

  A c c e p t e d M a n u s c r i p t 11

  effects of T on cultured human lung cancer cells, we profiled gene expression in A549 cells with or without T treatment. Androgen exposure for 24 h resulted in up-regulation of 126 transcripts, but only 62 of these were annotated as transcripts encoded by known genes (Supplemental data, Table

  than in cultured cells. Therefore, an intact organism (murine lung) is likely to express different gene sets than cells (A549 cells) under culture conditions.

Figure legends Fig. 1 .

 legends1 Figure legends

Fig. 2 .

 2 Fig. 2. Localization of AR, GR and hemoglobin in lung cells. A and B, Serial lung sections of male mice were immunostained with an antibody against AR (A) or GR (B) to show localization of AR to type II pneumocytes and bronchial epithelium, and the ubiquitous presence of GR. C and D, Influence of androgen treatment on AR localization. Castrated mice were given vehicle (C) or testosterone for 5 days (D). E and F, Influence of androgen treatment on hemoglobin immunostaining. Tissue sections from lungs of castrated (E) or androgen-treated (F) male mice were immunostained with antibody against hemoglobin //. Panels A and B were counterstained with hematoxylin. Scale bar = 50 µm.

Fig. 3 .

 3 Fig. 3. Immunoreactive AR in lung cancer specimens. Shown are examples of ARpositive sections from a human lung cancer tissue array comprising 80 spots from 40 lung cancer specimens and 4 non-neoplastic spots. The array was immunostained with AR antibody. The pictures show examples of immunoreactive spots from adenocarcinoma (A), squamous cell carcinoma (B), bronchioloalveolar carcinoma (C), small cell carcinoma (D), large cell carcinoma (E), and normal human lung tissue (F). Scale bar = 50 µm.

Fig. 4 .

 4 Fig. 4. Comparison of transcript numbers regulated by androgen and glucocorticoid in A549 cells. The cells were treated with 100 nM T, 1 µM Dex or vehicle for 24 h. Gene expression profiling was conducted as described in Materials and Methods. The figures show the overlap of the transcripts that were significantly regulated by either T or Dex.

Fig. 5 .

 5 Fig. 5. Loading of AR onto the TMPRSS2 and TAS2R50 enhancers in A549 cells upon androgen exposure. The cells were exposed to 100 nM or vehicle for 16 h. Precleared samples were immunoprecipitated with anti-AR-antibody. qRT-PCR was performed using primers for the TMPRSS2 and TAS2R50 enhancers and the bars represent relative foldchanges (+SEM) for T treatment in comparison to vehicle-treated samples (C) set at 1.0.PDGFRA refers to a control chromosomal site located at 57 kb down-stream of the PDGFRA gene.

  Fig.1

  Fig. 2

  Fig. 3

Fig. 5

 5 Fig. 5

Genes GO category p-value (GO) Gene Symbol Common Name Entrez ID Fold Change p-value

	oxygen transport negative regulation of apoptosis iron ion binding heme biosynthesis	3.59E-08 3.79E-03 5.93E-06 2.14E-04	Hbb-b1 Hbb-y Hbq1 Mb Angptl4 Tsc22d3 Fgf8 Hbb-b1 Rsad2 Fech Slc25a37 Hbb-y Hbq1 Mb Alas2 Fech	hemoglobin, beta adult major chain hemoglobin Y, beta-like embryonic chain hemoglobin, theta 1 myoglobin angiopoietin-like 4 TSC22 domain family 3 fibroblast growth factor 8 hemoglobin, beta adult major chain radical S-adenosyl methionine domain containing 2 ferrochelatase solute carrier family 25, member 37 hemoglobin Y, beta-like embryonic chain hemoglobin, theta 1 myoglobin aminolevulinic acid synthase 2, erythroid ferrochelatase M a n u s c r i p t	15129 15135 216635 17189 57875 14605 14179 15129 58185 14151 67712 15135 216635 17189 11656 14151	3.20 2.00 1.86 1.86 2.02 1.86 1.84 3.20 2.40 2.38 2.18 2.00 1.86 1.86 2.50 2.38	0.0276 0.0330 0.0028 0.0059 0.0075 0.0088 0.0182 0.0276 0.0007 0.0013 0.0034 0.0330 0.0028 0.0059 0.0023 0.0013
	Down-regulated Genes GO category DNA repair DNA recombination double-strand break repair response to stress	p-value (GO) 6.65E-03 9.19E-03 4.20E-03 1.73E-02 A c c e p t e d Gene Symbol Common Name Brca2 breast cancer 2 Msh6 mutS homolog 6 (E. coli) Pold3 polymerase (DNA-directed), delta 3, accessory subunit H2afx H2A histone family, member X Rev3l REV3-like, catalytic subunit of DNA polymerase zeta RAD54 like (S. cerevisiae) Brca2 breast cancer 2 Msh6 mutS homolog 6 (E. coli) H2afx H2A histone family, member X Brca2 breast cancer 2 H2afx H2A histone family, member X Brca2 breast cancer 2 C1s complement component 1, s subcomponent Hsp90ab1 heat shock protein 90kDa alpha (cytosolic), class B member 1 Msh6 mutS homolog 6 (E. coli) Pold3 polymerase (DNA-directed), delta 3, accessory subunit Fn1 fibronectin 1 Txnip thioredoxin interacting protein Lta4h leukotriene A4 hydrolase H2afx H2A histone family, member X	Entrez ID Fold Change p-value 12190 -2.08 0.0049 17688 -1.90 0.0269 67967 -1.90 0.0277 15270 -1.82 0.0219 19714 -1.81 0.0095 12190 -2.08 0.0049 17688 -1.90 0.0269 15270 -1.82 0.0219 12190 -2.08 0.0049 15270 -1.82 0.0219 12190 -2.08 0.0049 50908 -2.02 0.0044 15516 -1.90 0.0227 17688 -1.90 0.0269 67967 -1.90 0.0277 14268 -1.86 0.0171 56338 -1.85 0.0061 16993 -1.85 0.0158 15270 -1.82 0.0219
				Rev3l	REV3-like, catalytic subunit of DNA polymerase zeta RAD54 like (S. cerevisiae)	19714	-1.81	0.0095

Table 2 .

 2 Enriched gene ontology (GO) categories between intact male and female mouse lung Agilent Whole Mouse Genome expression microarray was used to compare gene-expression in the lung tissue of intact male and female mice. The GO categories are shown for transcripts that were differentially expressed (fold-change >1.8, t-test p value <0.05).

	Up-regulated genes
	GO category	p-value (GO)	Gene Symbol	Common Name

Table 3 .

 3 Genes differentially expressed by both T and Dex in A549 cellsAgilent Whole Human Genome expression microarray was used to compare gene-expression in A549 cells. Genes included in the lists were differentially expressed > 2-fold compared to vehicle (t-test p-value <0.05).

	Up-regulated Genes
	Gene Symbol	Common Name

Acknowledgments

We thank Saija Kotola, Johanna Iso-Oja and Anne Reijula for excellent technical assistance, Massimiliano Gentile for helping with the microarray analysis and Ismo Virtanen for helping with histological techniques. This work was supported by grants from the Academy of Finland, Biocentrum Helsinki, Sigrid Jusélius Foundation, Finnish Foundation for Cancer Research, Paulo Foundation, Oskar Öflund Foundation, Emil Aaltonen Foundation, Finnish Medical Foundation, Helsinki University Central Hospital, and European Union (CASCADE).


Manuel D Gahete

Jose Córdoba-Chacón

Roberto Salvatori

Justo P Castaño

Rhonda D Kineman

PhD Raul M Luque
email: raul.luque@uco.es

Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach

Keywords: Ghrelin O-Acyl Transferase (GOAT), mouse models (fasting, obesity, knockouts), stomach, pituitary, hypothalamus

A c c e p t e d M a n u s c r i p t Abstract Ghrelin acts as an endocrine link connecting physiological processes regulating food intake, body composition, growth, and energy balance. Ghrelin is the only peptide known to undergo octanoylation. The enzyme mediating this process, ghrelin O-acyltransferase (GOAT), is expressed in the gastrointestinal tract (GI; primary source of circulating ghrelin) as well as other tissues. The present study demonstrates that stomach GOAT mRNA levels correlate with circulating acylated-ghrelin levels in fasted and diet-induced obese mice. In addition, GOAT was found to be expressed in both the pituitary and hypothalamus (two target tissues of ghrelin's actions), and regulated in response to metabolic status. Using primary pituitary cell cultures as a model system to study the regulation of GOAT expression, we found that acylatedghrelin, but not desacyl-ghrelin, increased GOAT expression. In addition, growth-hormonereleasing hormone (GHRH) and leptin increased, while somatostatin (SST) decreased GOAT expression. The physiologic relevance of these later results is supported by the observation that pituitary GOAT expression in mice lacking GHRH, SST and leptin showed opposite changes to those observed after in vitro treatment with the corresponding peptides. Therefore, it seems plausible that these hormones directly contribute to the regulation of pituitary GOAT.

Interestingly, in all the models studied, pituitary GOAT expression paralleled changes in the expression of a dominant spliced-variant of ghrelin (In2-ghrelin) and therefore this transcript may be a primary substrate for pituitary GOAT. Collectively, these observations support the notion that the GI tract is not the only source of acylated-ghrelin, but in fact locally-produced des-acylated-ghrelin could be converted to acylated-ghrelin within target tissues by locally active GOAT, to mediate its tissue-specific effects.

Introduction

The primary source of circulating ghrelin is the stomach, where pro-ghrelin can be modified by the addition of an O-linked octanoyl side group added to its serine-3 residue, via the actions of ghrelin O-acyltransferase (GOAT) to produce acylated-proghrelin [START_REF] Kojima | Ghrelin is a growthhormone-releasing acylated peptide from stomach[END_REF][START_REF] Ariyasu | Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans[END_REF][START_REF] Garg | The ongoing saga of obestatin: is it a hormone?[END_REF][START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF][START_REF] Gutierrez | Ghrelin octanoylation mediated by an orphan lipid transferase[END_REF]Yang et al., 2008;[START_REF] Gomez | Expression and modulation of ghrelin O-acyltransferase in cultured chondrocytes[END_REF][START_REF] Sakata | Co-Localization of Ghrelin O-Acyltransferase (GOAT) and Ghrelin in Gastric Mucosal Cells[END_REF]. Then, prohormone convertase 1/3 (PC1/3) can further process pro-ghrelin (acylated or desacylated) to generate the mature 28-amino acid peptide ghrelin [START_REF] Garg | The ongoing saga of obestatin: is it a hormone?[END_REF]. Only a small proportion of circulating ghrelin is acylated, while the remaining is in the unmodified or des-acylated form, where both forms have biological effects [START_REF] Van Der Lely | Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin[END_REF][START_REF] Kojima | Ghrelin: structure and function[END_REF]. Acylated-ghrelin is the primary endogenous ligand for the growth hormone secretagogue receptor (GHSR), and it is now recognized that the acyl-ghrelin/GHSR system exerts actions on multiple tissues, many of them related to regulation of metabolic functions (for review, (van der Lely et al., 2004)). In particular, acylated-ghrelin works at the level of the hypothalamus as a potent orexigenic signal [START_REF] Kojima | Ghrelin: structure and function[END_REF], and at the level of the pituitary to modulate hormone release including augmentation of growth hormone (GH), adrenocorticotropin (ACTH) and prolactin release [START_REF] Kojima | Ghrelin: structure and function[END_REF][START_REF] Gahete | Understanding the multifactorial control of growth hormone release by somatotropes: lessons from comparative endocrinology[END_REF]. In addition, acylated ghrelin acts systemically to regulate glucose homeostasis and adiposity, where the deletion of the genes encoding ghrelin or its receptor prevents diet-induced obesity, improves insulin sensitivity and enhances glucose-stimulated insulin secretion (Yada et al., 2008).

Although the GI track is considered the major source of acylated ghrelin, other tissues express ghrelin, PC1/3 and GOAT [START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF][START_REF] Gutierrez | Ghrelin octanoylation mediated by an orphan lipid transferase[END_REF]Yang et al., 2008) (for review, (van der Lely et al., 2004)). Therefore, it is possible that locally produced non-acylated proghrelin might be converted to acylated-proghrelin within target tissues by locally-active GOAT, to mediate tissue specific effects. Local production of acyl-ghrelin is supported by a recent report demonstrating that acylated ghrelin can be produced by a thyroid A c c e p t e d M a n u s c r i p t and a pituitary cell line that express ghrelin, proconvertase 1/3 and GOAT if n-octanoic acid is available as a substrate [START_REF] Takahashi | Production of n-octanoyl-modified ghrelin in cultured cells requires prohormone processing protease and ghrelin O-acyltransferase, as well as n-octanoic acid[END_REF]. Given the fact that hypothalamus and pituitary are targets for acylated-ghrelin, coupled with the fact that these tissues also express ghrelin (for review, (van der Lely et al., 2004)), GOAT [START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF][START_REF] Gutierrez | Ghrelin octanoylation mediated by an orphan lipid transferase[END_REF]Yang et al., 2008) and PC1/3 [START_REF] Dong | Gene expression of proprotein convertases in individual rat anterior pituitary cells and their regulation in corticotrophs mediated by glucocorticoids[END_REF][START_REF] Nillni | Regulation of prohormone convertases in hypothalamic neurons: implications for prothyrotropin-releasing hormone and proopiomelanocortin[END_REF], it is possible that local production of acylated-ghrelin, as well as that produced by the GI tract, may play an important role in regulating this neuroendocrine axis in response to metabolic signals. Therefore, the goals of the current study were 1) to determine whether GOAT expression in the stomach correlates with circulating acylated-ghrelin levels in mice under conditions of metabolic stress (i.e., fasting, and obese mouse models), 2) to confirm if GOAT is expressed within the hypothalamus and pituitary and if expression levels are mediated by metabolic stress and 3) to determine whether primary regulatory factors for the pituitary-metabolic interface can directly regulate GOAT expression, by using primary mouse pituitary cell cultures as a model system.

Material and Methods

Animals and cell culture

All experimental procedures were approved by the Animal Care and Use Committees of the University of Cordoba, University of Illinois at Chicago and the Jesse Brown VA Medical Center. Mice were housed under standard conditions of light (12-h light, 12-h dark cycle; lights on at 0700 h) and temperature (22-24 C), with free access to tap water and food (standard rodent chow; LabDiet, St. Louis, MO; USA; Catalog no. 5008; fat, 17 kcal%, carbohydrate, 56 kcal%; protein, 27 kcal%). Leptin deficient ob/ob mice and their corresponding controls were purchased from Jackson laboratories, while growth hormone releasing hormone (GHRH), somatostatin (SST) and neuropeptide Y (NPY) knockout mice were bred in house. Original breeders were obtained from Dr. Ute Hochgeschwender (SST-KO), Dr. Richard D. Palmiter (NPY-KO) and Dr. Roberto Salvatori (GHRH-KO) as previously reported [START_REF] Alba | A mouse with targeted ablation of the growth hormone-releasing hormone gene: a new model of isolated growth hormone deficiency[END_REF][START_REF] Park | Expression analysis of hypothalamic and pituitary components of the growth hormone axis in fasted and streptozotocin-treated neuropeptide Y (NPY)-intact (NPY+/+) and NPY-knockout (NPY-/-) mice[END_REF]Luque et al., 2006;Luque and Kineman, 2006;Luque and Kineman, 2007;Luque et al., 2007b;Luque et al., 2007c). Mice were handled daily at least 1 week prior to A c c e p t e d M a n u s c r i p t euthanasia to acclimate them to personnel and handling procedures and were sacrificed by decapitation, without anesthesia, under fed conditions unless otherwise specified. Trunk blood was immediately mixed with MiniProtease inhibitor (Roche, Nutley, NJ) and placed on ice, centrifuged and plasma was stored at -80C until analysis of total-and acylated-ghrelin by ELISA (Linco, St. Charles, MO) following the manufacturer's instructions, including the addition of hydrochloric acid at a final concentration of 0.05N in order to prevent rapid desacylation of ghrelin after the plasma collection. Tissues (stomachs, pituitaries and hypothalami) were immediately frozen in liquid nitrogen and stored at -80ºC until further analysis of mRNA levels by quantitative real-time RT-PCR (qrtRT-PCR; see below).

Influence of metabolic stress on circulating total and acylated ghrelin and stomach, pituitary and hypothalamic GOAT mRNA levels

For these studies, serum and tissues previously processed and analyzed for other endpoints were used, where each study is listed below followed by a brief summary of the experimental design. Fasting -Ten week-old male C57Bl/6J mice were either fed ad libitum or fasted for 12h (food removed at 1900 h), 24h or 48h (food removed at 0700 h) (Luque et al., 2007c). An additional group of 8-9 week-old male C57Bl/6 x FVBN mice (n=5/group) were also fed ad libitum or fasted for 24h [START_REF] Kineman | Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress[END_REF]. For these and subsequent studies all tissues and blood were collected between 0700h-0900h. Diet-induced obesity (DIO) -Male C57Bl/6J mice (n = 6-7) were fed a low-fat (LFD; 10% kcal from fat) or a high-fat (HFD; 60% kcal from fat) diet starting at 4 week of age and killed at 20 wk (Luque and Kineman, 2006).

Leptin-deficient mice (ob/ob) -Blood and tissues were analyzed from 10 week old ob/ob mice and their littermates-controls (ob/?), as well as from ob/ob mice treated with vehicle (control 1), leptin (7d via osmotic mini-pumps released at a rate of 0.5 l/h, delivering a total of 15.6 g of leptin each day) or from a group of ob/ob mice pair-fed along with the leptin-treated animals that was used as an additional control (control 2) to match the food intake of leptin-treated mice (Luque and Kineman, 2006;Luque et al., 2007a) in order to differentiate between direct effects A c c e p t e d M a n u s c r i p t of leptin and those mediated indirectly by leptin-induced reduction in food intake and weight loss. GOAT mRNA levels by acylated-ghrelin, desacyl-ghrelin, GHRH, SST, leptin, NPY, insulin and IGF-I in

Direct regulation of

mouse primary pituitary cell cultures

To examine whether primary regulatory hormones for the pituitary-metabolic interface can directly control pituitary GOAT expression, pituitaries of 8-12 wk old male C57Bl6 mice obtained from "Centro de Instrumentación Científica" (University of Granada, Spain) or from Jackson Laboratories (Bar Harbor, ME, USA)) were enzymatically dispersed (n=3-5 pituitaries pooled/experiment, 6-7 separate experiments) into single cells and cultured (250,000 cells/well, 24-well plates) in serum containing α-MEM, as previously described (Luque et al., 2006;Luque and Kineman, 2006). After 24-48h of culture, media was removed and wells were washed in serum free media and subsequently treated with mouse acylated-ghrelin, desacyl-ghrelin, GHRH, NPY, insulin, IGF-I (10nM each), SST-14 (100nM) and mouse leptin (10ng/ml), for 24h (Sigma, Saint Louis, MO or Phoenix, Burlingame, CA; USA), and then total cellular RNA was extracted for determination of GOAT mRNA levels by qrtRT-PCR (see below). In order to determine if the direct actions of GHRH, SST, NPY or leptin could be important in maintaining in vivo expression of pituitary GOAT, total RNA obtained from pituitaries of 9-11 week old GHRH-knockout (KO) (B6129PF1/J), SST-KO mice (C57Bl/6J), NPY-KO mice (129/sv), leptin-KO (ob/ob; C57Bl/6J) and their respective littermate controls used in other studies [START_REF] Alba | A mouse with targeted ablation of the growth hormone-releasing hormone gene: a new model of isolated growth hormone deficiency[END_REF][START_REF] Park | Expression analysis of hypothalamic and pituitary components of the growth hormone axis in fasted and streptozotocin-treated neuropeptide Y (NPY)-intact (NPY+/+) and NPY-knockout (NPY-/-) mice[END_REF]Luque et al., 2006;Luque and Kineman, 2006;Luque and Kineman, 2007;Luque et al., 2007b;Luque et al., 2007c), were also evaluated from GOAT mRNA levels by qrtRTPCR.

RNA isolation and reverse transcription (RT)

Tissues and cell cultures were processed for recovery of total RNA using the Absolutely RNA RT-PCR Miniprep Kit with Deoxyribonuclease treatment (Stratagene, La Jolla, CA) as previously described (Luque et al., 2006;Luque and Kineman, 2006;Luque and Kineman, A c c e p t e d M a n u s c r i p t 2007; Luque et al., 2007c). The amount of RNA recovered was determined using the Ribogreen RNA quantification kit (Molecular Probes, Eugene, OR). Total RNA (1 g for whole tissue extract and 0.25 g for primary cell cultures) was reversed transcribed with enzyme and buffers supplied in the cDNA First Strand Synthesis kit (MRI Fermentas, Hanover, MD). cDNA was treated with ribonuclease H (1U; MRI Fermentas) and duplicate aliquots (1 l) were amplified by qrtRT-PCR, in which samples were run against synthetic standards to estimate mRNA copy number, as described below.

Quantification of GOAT and ghrelin mRNA levels by qrtRT-PCR

Details regarding the development, validation, and application of a qrtRT-PCR to measure expression levels of mouse transcripts, including ghrelin mRNA levels, have been reported previously (Luque et al., 2006;Luque and Kineman, 2006;[START_REF] Kineman | Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress[END_REF]Luque et al., 2007a;Luque and Kineman, 2007;Luque et al., 2007c). Specific primer sequences, GenBank accession numbers and product sizes for mouse GOAT, ghrelin and cyclophilin used in this study were as follows: GOAT (Mboat4; forward 5´-ATTTGTGAAGGGAAGGTGGAG-3´ and reverse 5´-CAGGAGAGCAGGGAAAAAGAG-3´, NM_001126314, 120bp); Ghrelin (forward 5´-TCCAAGAAGCCACCAGCTAA-3´ and reverse 5´-AACATCGAAGGGAGCATTGA-3´, NM_021488, 126bp) and cyclophilin (forward 5´-TGGTCTTTGGGAAGGTGAAAG-3´ and reverse 5´-TGTCCACAGTCGGAAATGGT-3´, NM_008907, 109bp). Primers were selected using Primer 3 software [START_REF] Rozen | Primer3 on the WWW for general users and for biologist programmers[END_REF] with selection parameters set to identify primer sets that: 1) span an intron (when possible), 2) differ by no more that 1 0 C in annealing temperature, 3) are at least 20bp in length, 4) have a GC content between 45-55%, but 5) exclude primers that may form primer-dimers.

Sequences of selected primers were used in BLAST (NCBI) searches to check for potential homology to sequences other than the designated target. Initial screening of primer efficiency using real-time detection was performed by amplifying 2-fold dilutions of RT products, where optimal efficiency was demonstrated by a difference of one cycle threshold between dilutions A c c e p t e d M a n u s c r i p t and a clear melting peak followed by a graded temperature-dependent dissociation to verify that only one product was amplified. The thermocycling profile consisted of one cycle of 95 C for 10 min, 40 cycles of 95 C for 30 sec, 61 C for 1 min, and 72 C for 30 sec. PCR products were then column-purified (QIAGEN, Valencia, CA) and sequenced to confirm target specificity.

After confirmation of primer efficiency and specificity, the concentration of purified products was determined using Molecular Probe's Picogreen DNA quantification kit, and PCR products were serial diluted to obtain standards containing 1, 10 1 ,10 2 , 10 3 , 10 4 , 10 5 , and 10 6 copies of synthetic template. Standards were then amplified by real-time PCR, and standard curves were generated using Stratagene Mx3000p software. The slope of a standard curve for each template examined was approximately 1, indicating that the efficiency of amplification of our primers was 100%, meaning that all templates in each cycle were copied. To determine the starting copy number of cDNA, RT samples were PCR amplified and the signal was compared with that of a specific standard curve of each transcript run on the same plate. In addition, total RNA samples that were not reversed transcribed and a no DNA control were run on each plate to control for genomic DNA contamination and to monitor potential exogenous contamination, respectively. Also, to control for variations in the amount of RNA used in the RT reaction and the efficiency of the RT reaction, mRNA copy number of the transcript of interest was adjusted by the mRNA copy number of cyclophilin A (used as housekeeping gene), where cyclophilin A mRNA levels did not significantly vary between experimental groups, within tissue type or treatment group (data not shown).

Data presentation and statistical analysis

Samples from all groups within an experiment were processed at the same time. The effects of fasting and leptin replacement were assessed by one-way ANOVA followed by a Newman-Keuls test for multiple comparisons, while the effects of obesity, genotype and the in vitro effects of acylated-ghrelin, desacyl-ghrelin, GHRH, SST, NPY, leptin, insulin and IGF-I were assessed by Student´s t-test. P < 0.05 was considered significant. All data are expressed as means ± SEM. The in vivo effects of fasting/obesity/genotype were obtained from a minimum

Results and Discussion

Quantification of GOAT mRNA levels in mouse stomachs, pituitaries and hypothalami

Using qrtRT-PCR, we found that GOAT mRNA levels were 5-fold greater in the stomach as compared to the pituitary and hypothalamus (Table 1). These results are consistent with other reports showing relative expression levels of GOAT in both human [START_REF] Gutierrez | Ghrelin octanoylation mediated by an orphan lipid transferase[END_REF] and rodent [START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF]Yang et al., 2008;[START_REF] Sakata | Co-Localization of Ghrelin O-Acyltransferase (GOAT) and Ghrelin in Gastric Mucosal Cells[END_REF] tissues.

Interestingly, the expression level of GOAT transcripts in the mouse tissues analyzed in the current study (table 1) paralleled the mRNA levels of ghrelin in the same tissues (Stomach pituitary = hypothalamus), as previously reported [START_REF] Kineman | Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress[END_REF].

Since it has been previously reported that the enzyme prohormone convertase 1/3 which is required for the conversion of preprohormones, including preproghrelin to its 28 amino acid form, is also expressed in the pituitary and hypothalamus [START_REF] Dong | Gene expression of proprotein convertases in individual rat anterior pituitary cells and their regulation in corticotrophs mediated by glucocorticoids[END_REF][START_REF] Nillni | Regulation of prohormone convertases in hypothalamic neurons: implications for prothyrotropin-releasing hormone and proopiomelanocortin[END_REF], as well as the GI tract [START_REF] Macro | Identification and expression of prohormoneconverting enzymes in the rat stomach[END_REF], it is possible that the source of acylated-ghrelin within the pituitary and hypothalamus may include locally produced ghrelin, in addition to that found in the circulation. Therefore, the current study compared the impact of metabolic stress (fasting and obesity) on circulating ghrelin (acylated and total) levels, as well as GOAT and ghrelin mRNA levels in the stomach, pituitary and hypothalamus. Specifically, Figure 1 illustrates the impact of fasting, diet-induced obesity (DIO) and obesity caused by leptin deficiency (ob/ob mice) on circulating acylated-and total-ghrelin levels and stomach GOAT mRNA levels (Panels A, B and C, respectively), as well as pituitary and hypothalamic GOAT mRNA levels (Panels D, E and F, respectively). It should be noted that, in some cases we have previously published the regulation of stomach, pituitary and hypothalamic ghrelin mRNA A c c e p t e d M a n u s c r i p t levels in these model systems [START_REF] Kineman | Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress[END_REF]Luque et al., 2007a;Luque et al., 2007c).

These results are summarized in Figure 1 as the relative changes in ghrelin mRNA levels (=, ↑ , ↓ ), as compared to controls, as shown below each graph of tissue-specific GOAT expression.

Effects of nutrient deprivation on GOAT/ghrelin axis.

As shown in Figure 1, panel A, fasting did not alter circulating levels of total-ghrelin or stomach ghrelin expression, but did increase circulating acylated-ghrelin (12 and 24h) and stomach GOAT mRNA levels (24h). The impact of 24h fasting on stomach GOAT mRNA levels was confirmed in tissues taken from an independent set of male mice from another study [START_REF] Kineman | Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress[END_REF], using two different primer sets for amplification of the GOAT transcripts (supplemental Fig. 1). These results differ from those of a recent report by Kirchner et al., [START_REF] Kirchner | GOAT links dietary lipids with the endocrine control of energy balance[END_REF] showing that fasting (12h, 24h and 36h) significantly increased total-ghrelin levels and tended to increase acylated-ghrelin levels, but unexpectedly suppressed stomach GOAT mRNA in C57Bl/6 male mice. It is possible that such differences may be related to the time of food withdrawal, time of sample collection, type of standard rodent chow provided and/or the method of euthanasia which were not clearly defined in that study. In fact, Kirchner et al., [START_REF] Kirchner | GOAT links dietary lipids with the endocrine control of energy balance[END_REF] observed a diurnal pattern of circulating ghrelin and stomach ghrelin/GOAT mRNA levels, as well as an association between stomach GOAT expression and the type of dietary lipids supplied. It is also possible that the differences observed are related to the analytical techniques applied, where circulating ghrelin levels were assessed by commercial assays in the current study, but in the case of Kirchner et al., [START_REF] Kirchner | GOAT links dietary lipids with the endocrine control of energy balance[END_REF], were assessed by MALDI-TOF mass spectrometry of immunoprecipitated ghrelin.

Finally, with respect to GOAT mRNA levels, although both the current study and that of [START_REF] Kirchner | GOAT links dietary lipids with the endocrine control of energy balance[END_REF] used real-time RT-PCR, the primer sets used and the characteristics and properties of the primers were not the same (length of primers, GC content, etc.). Nonetheless, our current observations are consistent with other reports showing a clear rise in acylated-ghrelin levels in fasted humans and other species (for review: [START_REF] Casanueva | Ghrelin: the link connecting growth with metabolism and energy homeostasis[END_REF][START_REF] Gottero | Ghrelin: a link between eating disorders, obesity and reproduction[END_REF][START_REF] Kojima | Ghrelin: structure and function[END_REF] [START_REF] Perreault | Resistance to the orexigenic effect of ghrelin in dietary-induced obesity in mice: reversal upon weight loss[END_REF]Luque et al., 2006;Luque et al., 2007c;Zizzari et al., 2007), as well as an increase in stomach GOAT mRNA levels in rats subjected to 21-days of caloric-restriction [START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF]. However, it should be noted that like Kirchner et al., Liu et al. failed to detect a rise in circulating acylated ghrelin in fasted humans [START_REF] Liu | Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men[END_REF]. It should also be emphasized that in the current study the fastinginduced rise in acylated-ghrelin was observed at 12 and 24h, but began to fall after 48h, while the fasting-induced rise in GOAT mRNA was only observed at 24h. These results suggest that an increase in GOAT expression levels in the stomach may only in part contribute to the rise in circulating acyl-ghrelin, where enhanced GOAT activity and/or substrate availability may precede that process. However, after 48h of fasting circulating acylated-ghrelin levels and GOAT mRNA levels did not differ from controls (Fig. 1, panel A), where 48h of fasting in a mouse represent a severe catabolic state (Luque et al., 2007c). This observation is consistent with reports showing that the proportion of circulating acylated-ghrelin falls after long-term fasting in humans (61.5h-fasting) [START_REF] Liu | Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men[END_REF] and rats (48h-fasting) [START_REF] Toshinai | Upregulation of Ghrelin expression in the stomach upon fasting, insulininduced hypoglycemia, and leptin administration[END_REF][START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF] and with the recent observation indicating that 48h of fasting did not significantly alter stomach GOAT mRNA expression in rats [START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF][START_REF] Takahashi | Production of n-octanoyl-modified ghrelin in cultured cells requires prohormone processing protease and ghrelin O-acyltransferase, as well as n-octanoic acid[END_REF]. Taken together our current results suggest that changes in stomach GOAT expression may contribute in part to fasting-induced changes in circulating acylated-ghrelin levels, a hypothesis previously put forth by other laboratories [START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF][START_REF] Gualillo | Introducing GOAT: a target for obesity and anti-diabetic drugs?[END_REF]. However, given the only other report examining this relationship in mice gave different results [START_REF] Kirchner | GOAT links dietary lipids with the endocrine control of energy balance[END_REF], and the fact that there are many potential levels of GOAT regulation (transcriptional, translational, enzyme activity level, substrate availability), it will be important for other laboratories to validate and confirm these findings.

Effects of obesity on GOAT/ghrelin axis.

As shown in Figure 1, panel B, diet-induced obesity tended (50% reduction, p=0.13) to suppress total ghrelin levels in mice without altering acylated-ghrelin levels, consistent with a significant decline in stomach ghrelin expression, while GOAT mRNA levels remained A c c e p t e d M a n u s c r i p t unchanged. In contrast, obesity as a result of leptin-deficiency (ob/ob mice, Fig. 1 panel C) led to a significant decline in circulating acylated-ghrelin levels (and a downward trend in totalghrelin levels), changes which were accompanied by a significant decrease in stomach ghrelin but not GOAT mRNA levels, similar to DIO mice. This is the first report to explore the impact of DIO on stomach GOAT and ghrelin expression, while a previous study showed similar results for stomach GOAT and ghrelin mRNA levels in ob/ob mice [START_REF] Kirchner | GOAT links dietary lipids with the endocrine control of energy balance[END_REF]. In addition, others have reported a significant decline in total-or desacyl-ghrelin in DIO [START_REF] Ueno | Blunted metabolic response to fasting in obese mice[END_REF] and obese Ay [START_REF] Nonogaki | Hyperphagia alters expression of hypothalamic 5-HT2C and 5-HT1B receptor genes and plasma des-acyl ghrelin levels in Ay mice[END_REF] mice and a reduction in total-and acylatedghrelin levels in obese humans [START_REF] Casanueva | Ghrelin: the link connecting growth with metabolism and energy homeostasis[END_REF][START_REF] Gottero | Ghrelin: a link between eating disorders, obesity and reproduction[END_REF][START_REF] Ghigo | Ghrelin: more than a natural GH secretagogue and/or an orexigenic factor[END_REF][START_REF] Kojima | Ghrelin: structure and function[END_REF], where DIO-induced alterations in acylated-ghrelin levels may be dependent on the time of day sampled [START_REF] Perreault | Resistance to the orexigenic effect of ghrelin in dietary-induced obesity in mice: reversal upon weight loss[END_REF]. Taken together these results indicate that the decline in circulating total-ghrelin levels in the obese state could be in part due to a decline in stomach ghrelin gene expression, however alterations in circulating acylated-ghrelin may be related to factors independent of GOAT expression, such as the rate of degradation of acylated-ghrelin, GOAT enzyme activity levels or substrate (ghrelin or fatty acid) availability.

Impact of metabolic stress (fasting and obesity) on hypothalamic and pituitary GOAT expression.

As shown in Figure 1, panel D, fasting also enhanced expression of GOAT at both the hypothalamic [at 48h (p=0.0016)] and pituitary [24h (p=0.017) and 12h (p=0.051)] level.

Interestingly, it should be noted that the timing of the GOAT mRNA rise was different from that observed in the stomach (Fig. 1,panel A). Conversely, DIO (Fig. 1, panel E) and obesity caused by leptin-deficiency (Fig. 1, panel F) did not alter hypothalamic GOAT expression, but did result in a significant decline in pituitary GOAT mRNA levels. As in the stomach, hypothalamic and pituitary GOAT expression did not always parallel local changes in ghrelin expression.

Taken together, these results suggest local production (ghrelin gene expression) and/or modification (via GOAT gene expression) of ghrelin may contribute to regulation of A c c e p t e d M a n u s c r i p t hypothalamic and pituitary function independent of circulating ghrelin levels produced by the stomach which would be supported by a recent study indicating that various cell lines, including the mouse pituitary cell line AtT20, can produce acylated-ghrelin when both ghrelin and GOAT are present in the cells and when substrate (fatty acid) is available in the culture medium [START_REF] Takahashi | Production of n-octanoyl-modified ghrelin in cultured cells requires prohormone processing protease and ghrelin O-acyltransferase, as well as n-octanoic acid[END_REF] 3.5. Direct regulation of GOAT mRNA levels by ghrelin, desacyl-ghrelin, GHRH, SST, leptin, NPY, insulin and IGF-I in mouse primary pituitary cell cultures.

Given the dynamic changes in pituitary GOAT expression in both fasting (upregulation; Fig. 1,panel D) and obesity (down-regulation; Fig. 1, panel E and F), we used primary mouse pituitary cell cultures as a model system to explore which central or systemic factors might mediate these changes. First, to confirm that mouse primary pituitary cells maintain differentiated function (ie. GOAT expression) after dispersion and culture, the absolute mRNA levels (copy numbers/0.05 g total RNA) of GOAT were compared between whole tissue extracts and extracts prepared from pituitary cultures 24h after incubation in serum-free media, and the results are shown in Table 1. Transcript levels did not significantly vary between in vivo and in vitro samples, indicating that the cell preparation and culture conditions were appropriate to maintain GOAT expression. Based on reports from our laboratory and others showing that the levels and/or actions of ghrelin, GHRH and NPY are up-regulated in fasting [START_REF] Henry | Chronic food-restriction alters the expression of somatostatin and growth hormone-releasing hormone in the ovariectomised ewe[END_REF][START_REF] Park | Expression analysis of hypothalamic and pituitary components of the growth hormone axis in fasted and streptozotocin-treated neuropeptide Y (NPY)-intact (NPY+/+) and NPY-knockout (NPY-/-) mice[END_REF]Luque et al., 2006;Luque et al., 2007c;[START_REF] Kirchner | GOAT links dietary lipids with the endocrine control of energy balance[END_REF] and down-regulated in obesity [START_REF] Takahashi | Production of n-octanoyl-modified ghrelin in cultured cells requires prohormone processing protease and ghrelin O-acyltransferase, as well as n-octanoic acid[END_REF][START_REF] Ahmad | Obesity-associated decrease in growth hormone-releasing hormone gene expression: a mechanism for reduced growth hormone mRNA levels in genetically obese Zucker rats[END_REF][START_REF] Maccario | Assessment of GH/IGF-I axis in obesity by evaluation of IGF-I levels and the GH response to GHRH+arginine test[END_REF][START_REF] Lin | Leptin receptor, NPY, POMC mRNA expression in the diet-induced obese mouse brain[END_REF][START_REF] Perreault | Resistance to the orexigenic effect of ghrelin in dietary-induced obesity in mice: reversal upon weight loss[END_REF]Luque and Kineman, 2006;[START_REF] Nonogaki | Hyperphagia alters expression of hypothalamic 5-HT2C and 5-HT1B receptor genes and plasma des-acyl ghrelin levels in Ay mice[END_REF]Luque et al., 2007a) while those for SST, leptin, insulin and IGF-I are down-regulated in fasting [START_REF] Henry | Chronic food-restriction alters the expression of somatostatin and growth hormone-releasing hormone in the ovariectomised ewe[END_REF][START_REF] Frystyk | Free insulin-like growth factors --measurements and relationships to growth hormone secretion and glucose homeostasis[END_REF][START_REF] Park | Expression analysis of hypothalamic and pituitary components of the growth hormone axis in fasted and streptozotocin-treated neuropeptide Y (NPY)-intact (NPY+/+) and NPY-knockout (NPY-/-) mice[END_REF]Luque et al., 2006;[START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF] and up-regulated in obesity (Zhou et al., 1997;[START_REF] Frystyk | Free insulin-like growth factors --measurements and relationships to growth hormone secretion and glucose homeostasis[END_REF]Luque and Kineman, 2006;Luque et al., 2007b;[START_REF] Luque | Role of endogenous somatostatin in regulating GH output under basal conditions and in response to metabolic extremes[END_REF], we incubated primary pituitary cell cultures with these hormones for 24h and measured the impact on GOAT mRNA levels. As shown in Figure 2, top panel, acylated-ghrelin clearly stimulated GOAT expression compared to vehicle-treated A c c e p t e d M a n u s c r i p t controls (set at 100%). In order to confirm that this effect on GOAT mRNA levels was directly exerted by acylated-ghrelin and not by desacyl-ghrelin (as result of a des-octanoylation of acylated-ghrelin in the pituitary cell cultures after 24h of incubation), pituitary cell cultures were treated with desacyl-ghrelin for 24h. The results clearly indicate that treatment with desacylghrelin did not alter GOAT mRNA levels. In addition, as shown in figure 2 (top panel), GHRH and leptin stimulated and SST inhibited pituitary GOAT expression, while NPY, insulin and IGF-I had no effect, compared to vehicle-treated controls (set at 100%). In order to determine, if these direct actions might also be important in maintaining pituitary GOAT expression in vivo, we examined pituitary expression of GOAT in available mouse models that lack GHRH, SST, NPY and leptin. Accordingly, we found an opposite effect of GHRH-KO, SST-KO and leptin-KO on pituitary GOAT expression, as compared to that observed after in vitro treatment with the corresponding hormone (Fig. 2, bottom panel). It is also interesting to note that when the impact of leptin replacement was examined in the ob/ob model (Luque et al., 2007a), we observed that pituitary and stomach GOAT mRNA levels were greater than that observed in pair-fed controls (Supplemental Figure 2), which is consistent with the report of Gonzalez et al., [START_REF] Gonzalez | Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa[END_REF] showing that administration of exogenous leptin markedly increased expression levels of GOAT in the stomach of fasted rats. Taken together these results indicate that acylated-ghrelin may in fact regulate its own production at the level of the pituitary by increasing the expression of GOAT. In addition, these results clearly demonstrate that GHRH, SST and leptin are key regulators of pituitary GOAT expression, an observation which may in part explain changes in pituitary GOAT expression observed in response to metabolic extremes.

Intriguingly, the substrate of GOAT activity in the mouse pituitary may not be limited to native ghrelin being locally produced within the pituitary, because our laboratory has identified a spliced mRNA ghrelin variant containing exon2, intron2 and exon3, but lacking exon1, 4 and 5 (In2-ghrelin variant; [START_REF] Kineman | Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress[END_REF]) which is expressed at higher levels than the native ghrelin transcript in the pituitary (139 24 copies for In2-ghrelin variant versus 18 3 copies for native-ghrelin/0.05µg RNA). If this variant is translated, it would encode a protein that would include the first 36 amino acid of the pre-proghrelin, where residues 24-28 represent the A c c e p t e d M a n u s c r i p t first five amino acids of mature ghrelin and thus contain the Ser-3 acylation site. It is interesting to note that when we compared the impact of metabolic stress (fasting and DIO) or GHRH, SST and leptin deficiency on pituitary expression of GOAT, native ghrelin and In2-ghrelin, the levels of GOAT expression paralleled those of In2-ghrelin variant but not native ghrelin, in all cases (summarized in Table 2, and details presented in Supplemental Figure 3). Based on these observations it seems reasonable to propose that the In2-ghrelin variant could be a primary substrate for GOAT in the anterior pituitary gland.

Although more experiments are required to fully elucidate the physiologic significance of our findings in the pituitary, our data give credence to the possibility that local production and/or modification of ghrelin (desacylated to acylated via GOAT gene expression) may contribute to the fine regulation of pituitary function in response to metabolic stress. For example, circulating GH levels are known to rise in response to fasting in the mouse, and this is associated with an increase in pituitary expression of GH, GHRH-R and GHS-R and a decline in SST receptor expression (rat and mouse; (Luque et al., 2007a;[START_REF] Luque | Role of endogenous somatostatin in regulating GH output under basal conditions and in response to metabolic extremes[END_REF]). The acute rise in systemic acylated-ghrelin, in combination with an increased sensitivity of the pituitary to ghrelin and GHRH actions, might initiate local production of acylated-ghrelin by augmentation of ghrelin and GOAT expression, thereby acting as a positive ultrashort feedback loop to enhance or facilitate GH release. In contrast, GH output is suppressed in obesity, and effect which is believed to be due in part to enhanced SST tone [for review see [START_REF] Luque | Role of endogenous somatostatin in regulating GH output under basal conditions and in response to metabolic extremes[END_REF]] and a decline in pituitary of GHRH-R, GHS-R and GH (Luque and Kineman, 2006;[START_REF] Luque | Role of endogenous somatostatin in regulating GH output under basal conditions and in response to metabolic extremes[END_REF]. In this context, our data would support the idea that part of the negative effects of SST on somatotrope function could include direct suppression of GOAT expression and a decline of locally produced acylated-ghrelin, further contributing to the decline in GH release observed with weight gain.

In summary, we report herein a series of novel analyses on the regulation of the mouse GOAT/ghrelin system at the circulating-stomach-pituitary-hypothalamic levels by energy status and relevant metabolic cues in the mouse. By applying a combination of studies involving in vivo models (fasting, obesity and knockout mice) and primary pituitary cell cultures, our results [START_REF] Kineman | Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress[END_REF]Luque et al., 2007a;Luque et al., 2007c).

GOAT mRNA

  per group. Results from in vitro studies were obtained from 3-7 separate, independent experiments (3-5 wells/treatment) carried out in different days and with different cells preparation. Endpoints displaying heterogeneity of variance were log transformed prior to analysis. All statistical analyses were performed using the GB-STAT software package (Dynamic Microsystems, Inc. Silver Spring, MD, USA).

  ; Williams and Cummings, A c c e p t e d M a n u s c r i p t 2005; Cummings, 2006)), including mice

  The authors have nothing to disclose.A c c e p t e d M a n u s c r i p t

*

  Differences observed only in females.

Figure 1 :Figure 2 :

 12 Figure 1: Circulating total-and acylated-ghrelin and stomach, hypothalamus and pituitary GOAT mRNA levels in: mice fed ad libitum or fasted for 12-, 24-, and 48-h (Panel A and D ), mice fed a low-fat diet (LFD control group) or high-fat diet (HFD obese group) (Panel B and E) and lean controls ( /?) and obese (ob/ob) mice (Panel C and F). Values are shown as mean ± SEM (n=5-8 mice/group) and expressed as percent of the respective control group (set at 100%). Group means that do not share a common letter (a,b) significantly differ (p<0.05; Panel A and D). Asterisks (*, p<0.05, **, p<0.01) indicate values that significantly differ from lean control LFD or  /?values. The tissue-specific regulation of ghrelin mRNA levels in these mice models as previously reported[START_REF] Kineman | Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress[END_REF] Luque et al., 2007a; Luque et al., 2007c) are shown below each graph of tissue-specific GOAT expression and are provided as the relative changes (=, ↑ , ↓ ) as compared to controls.

  Figure

Table 1 :

 1 Absolute cDNA copy number/0.05 g total RNA of GOAT gene transcripts in the stomach, pituitary and hypothalamus of male C57Bl/6 mice, as determined by quantitative realtime RT-PCR.

	GOAT mRNA copy

Table A

 A

	M a n u s c r i p t
	c c e p t e d

Table 2 :

 2 Relative effect of metabolic condition (fasting, obesity) or inactivating mutations in growth hormone releasing hormone (GHRH), somatostatin (SST) or leptin genes on pituitary GOAT, ghrelin and In2-ghrelin variant mRNA levels, as determined by quantitative real-time RT-PCR. This summary represents data presented in the current report, combined with previously published reports

Acknowledgements

The authors would like to thank Dr. Ute Hochgeschwender (Oklahoma Medical Research Foundation, Oklahoma City, OK) for the SST knockout mice, Dr. Richard D. Palmiter (Howard Hughes Medical Institute, University of Washington, Seattle Seattle, WA) for the NPY knockout

Funding

This work is supported in part by grants from Programa Nacional de becas de FPU (FPU-AP20052473, to MDG) and Programa Ramon y Cajal del Ministerio de Educación y Ciencia, Spain (RYC-2007-00186, to RML); Ayudas predoctorales de formacion en investigacion en salud del Fondo de Investigación sanitaria del Instituto de Salud Carlos III (FI06/00804; to JCC) and by grants from Junta de Andalucía BIO-0139 and CTS-01705 and Ministerio de Ciencia e Innovacion/FEDER BFU2007-60180/BFI, BFU2008-01136/BFISpain, and NIDDK 30677 and the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development Merit Award (to RDK). CIBER is an initiative of Instituto de Salud Carlos III (Ministerio de Sanidad, Ministerio de Ciencia e Innovación, Spain).

A c c e p t e d M a n u s c r i p t further characterize the impact of changes in body energy stores on the expression of GOAT in different tissues and define the role of key regulators, such as acylated-ghrelin itself, GHRH, SST and leptin on the control of GOAT expression in the mouse. The fact that GOAT mRNA levels are oppositely regulated in extreme metabolic states (fasting, DIO) and its regulation is tissue-dependent suggests that this enzyme may be of biological relevance in coordinating the neuroendocrine response to metabolic stress. Although much work remains to be done to fully understand how GOAT fits into the control of energy homeostasis, our results reinforce the contention that GOAT/acylated-ghrelin system could operate as a novel molecular conduit for the integration of energy balance, metabolism and pituitary function.