Effects of dihydrotestosterone on adipose tissue measured by serial analysis of gene expression

in Journal of Molecular Endocrinology

Intra-abdominal fat accumulation is related to several diseases, especially diabetes and heart disease. Molecular mechanisms associated with this independent risk factor are not well established. Through the serial analysis of gene expression (SAGE) strategy, we have studied the transcriptomic effects of castration and dihydrotestosterone (DHT) in retroperitoneal adipose tissue of C57BL6 male mice. Approximately 50 000 SAGE tags were isolated in intact and gonadectomized mice, as well as 3 and 24 h after DHT administration. Transcripts involved in energy metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, malic enzyme supernatant, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase and monoglyceride lipase, were upregulated by DHT. Transcripts involved in adipogenesis, and cell cycle and cell shape organization, such as DDX5, C/EBPα, cyclin I, procollagen types I, III, IV, V and VI, SPARC and matrix metalloproteinase 2, were upregulated by DHT. Cell defense, division and signaling, protein expression and many novel transcripts were regulated by castration and DHT. The present results provide global genomic evidence for a stimulation of glycolysis, fatty acids and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation, by DHT. The novel transcripts regulated by DHT may contribute to identify new mechanisms involved in the action of sex hormones and their potential role in obesity.

Abstract

Intra-abdominal fat accumulation is related to several diseases, especially diabetes and heart disease. Molecular mechanisms associated with this independent risk factor are not well established. Through the serial analysis of gene expression (SAGE) strategy, we have studied the transcriptomic effects of castration and dihydrotestosterone (DHT) in retroperitoneal adipose tissue of C57BL6 male mice. Approximately 50 000 SAGE tags were isolated in intact and gonadectomized mice, as well as 3 and 24 h after DHT administration. Transcripts involved in energy metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, malic enzyme supernatant, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase and monoglyceride lipase, were upregulated by DHT. Transcripts involved in adipogenesis, and cell cycle and cell shape organization, such as DDX5, C/EBPα, cyclin I, procollagen types I, III, IV, V and VI, SPARC and matrix metalloproteinase 2, were upregulated by DHT. Cell defense, division and signaling, protein expression and many novel transcripts were regulated by castration and DHT. The present results provide global genomic evidence for a stimulation of glycolysis, fatty acids and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation, by DHT. The novel transcripts regulated by DHT may contribute to identify new mechanisms involved in the action of sex hormones and their potential role in obesity.

Keywords:

Introduction

Obesity is a growing epidemic in many countries, especially in sedentary populations (Seidell 2000). Important health disorders, such as cardiovascular diseases, atherosclerosis, diabetes mellitus and cancer (Bray 1985, Kissebah et al. 1989, Calle et al. 2003), are associated with obesity. In fact, there is a correlation between the distribution of fat accumulation and health disorders (Despres et al. 1990, Vague 1956, Sheehan & Jensen 2000). Sex hormones partly define the localization of fat accumulation. Men tend to gain fat in the abdominal region while women accumulate fat mainly in the gluteal-femoral area (Sjostrom et al. 1972). The male pattern of fat accumulation is more closely associated with health disorders (Vague 1956, Sheehan & Jensen 2000). A link may thus exist between androgen levels, fat accumulation and fat distribution. However, until now, the global molecular effects of androgens on adipose tissue have not been elucidated.

With the advent of DNA microarrays (Schena et al. 1995) and serial analysis of gene expression (SAGE) (Velculescu et al. 1995), new possibilities arose for large-scale transcriptome analysis and comparison. Both techniques enable characterization of the transcriptome under multiple experimental conditions. DNA microarrays are restricted to known sequences and have some limitations in quantification (Novak et al. 2002). However, the SAGE method is highly quantitative and does not require previous knowledge of the sequences under study. This powerful strategy allows us to characterize the entire transcriptome and perhaps discover novel genes (Velculescu et al. 1997, St-Amand et al. 2001).

We have already presented the transcriptome of normal adipose tissue in mice (Bolduc et al. 2004). In the present study, using the SAGE strategy, we show the effects of castration and dihydrotestosterone (DHT), the most potent androgen, on adipose tissue of male mice. The transcripts involved in fat metabolism are discussed, as well as many transcripts involved in various functions modulated by androgens in this tissue. These findings constitute the first step towards a precise understanding of the molecular mechanisms involved in the physiological effects of androgens on adipose tissue.

Materials and methods

Sample preparation

Retroperitoneal adipose tissue was obtained from 10 male C57BL6 12–14-week-old mice per group, purchased from Charles River Canada Inc (St Constant, Canada). The animals had access to Lab Rodent Diet No. 5002 and water ad libitum. A sham gonadectomy was performed 7 days prior to organ collection for the intact group, while gonadectomy was performed at the same time for the three gonadectomized (GDX) groups. DHT (0.1 mg) was injected 3 h (DHT3h) and 24 h (DHT24h) prior to killing in groups 3 and 4. The dose of DHT selected was the smallest dose that could restore the prostate weight of GDX mice to the level of intact mice. The control groups (intact and GDX) received vehicle solution (0.4% (w/v) Methocel A15LV Premium/ 5% ethanol) instead of DHT. All animal experimentation was conducted in accord with accepted standards of humane animal care. The retroperitoneal adipose tissue was dissected between 0900 and 1215 h. The samples from all mice of the same group were pooled to eliminate interindividual variations and to extract sufficient amount of mRNA. The tissues were stored at − 80 °C until RNA extraction.

Transcriptome analysis

The SAGE method was performed as previously described (Velculescu et al. 1995, 1997, Kenzelmann & Muhlemann 1999, St-Amand et al. 2001). Polyadenylated RNA was extracted with the mRNA direct kit (Dynal, Oslo, Norway), annealed with the biotin-5′-T18-3′ primer and converted to cDNA with the cDNA synthesis kit (Invitrogen). The resulting cDNA library was digested with NlaIII (anchoring enzyme), and the 3′ restriction fragments were isolated with streptavidin-coated magnetic beads (Dynal) and separated into two populations. Each population was ligated to one of the two annealed linker pairs and extensively washed to remove unligated linkers. The tag beside the most 3′ NlaIII restriction site (CATG) of each transcript was released by digestion with BsmFI (tagging enzyme). The blunting kit from Takara Co. (Otsu, Japan) was used for the blunting and ligation of the two tag populations. The resulting ligation products containing the ditags were amplified by PCR with an initial denaturation step of 1 min at 95 °C, followed by 28 cycles of 20 s at 94 °C, 20 s at 60 °C and 2 s at 72 °C with 27 bp primers (St-Amand et al. 2001). Due to the low mRNA content of adipose tissue, a second PCR amplification was performed on the ditags for 14 cycles in order to enhance the size of the SAGE library without affecting the quantitative information required for group comparisons (Virlon et al. 1999). Each amplification was followed by acrylamide gel purification of the ditags. Finally, large-scale PCR was performed for eight cycles. The PCR product was digested with NlaIII, and the band containing the ditags was extracted from the acrylamide gel. The purified ditags were self-ligated to form concatemers. The concatemers of 500–1800 bp were isolated by agarose gel. The resulting DNA fragments were ligated into the SphI site of pUC19 and cloned into UltraMAX DH5αFT (Invitrogen). White colonies were screened by PCR to select long inserts for automated sequencing.

Bioinformatic analysis

All SAGE tag sequences were deposited in the GEO database at the National Centre for Biotechnology Information (NCBI). Sequence files were analyzed by the SAGEana program, a modification of SAGEparser (ftp://ftp.pbrc.edu/public/eesnyder/SAGE/). Tags corresponding to linker sequences were discarded, and duplicate concatemers were counted only once. Identification of the transcripts was obtained by matching the 15 bp (CATG+11 bp tags) with the UniGene and GenBank databases. The matching procedure used was very restrictive since, in order to avoid the possibility of sequencing errors in the expressed sequence tags (EST) database, we did not consider the matches that were identified only once among the numerous sequences of an UniGene cluster. Indeed, the chance of matches with EST containing sequencing errors drops dramatically when at least two EST are identified in a UniGene cluster for a given tag sequence. A minimum of one EST with a known polyA tail had to be in the UniGene cluster to identifiy the last NlaIII site on the corresponding cDNA. Classification of the genes was based upon the updated information of the genome directory (Adams et al. 1995) found at the TIGR website (www.tigr.org/). To analyze the promoter sequences for the presence of hormone-responsive elements (HRE), the 2 kb upstream regions of the annotated transcription start of the differentially expressed transcripts were extracted from the mouse genome at NCBI (build 32, version 1). With a Perl script, the promoter sequences were parsed to find the occurrence and positions of the sequences TGTTCT and AGAACA, which are present in more than one natural androgen-responsive elements (Nelson et al. 1999). When the genes were on the minus strand of the mouse genome, the downloaded sequences were transformed into their reverse-complement before the parsing procedure.

Statistical analysis

We used the comparative count display (CCD) test to identify the transcripts that were differentially expressed significantly (P≤0.05) between the groups with more than a twofold change. The CCD test makes a key-by-key comparison of two key-count distributions by generating a probability that the frequency of any key in the distribution differs by more than a given fold factor from the other distribution. This statistical test has been described elsewhere (Lash et al. 2000). The data are normalized to 50 000 tags in order to facilitate visual comparison in the tables.

Results

Four libraries (intact, GDX, GDX+DHT3h and GDX+DHT24h) were generated to characterize the effects of castration and DHT on adipose tissue transcriptome. Approximately 50 000 tags were sequenced in each group for a total of 192 431 tags. Thus, 61 931 different transcript species were detected. The majority of the tags sequenced represent novel transcripts. However, most of the transcript species expressed at high levels correspond to transcripts of known genes (Table 1). The 196 well-characterized transcripts differentially expressed at a significant level (P≤0.05) are presented in Tables 2–8, according to their functions. There were 117 transcripts upregulated by DHT, 69 transcripts downregulated by DHT and 10 transcripts regulated by castration.

Genes involved in the glycolysis pathway, such as the aldolase 1 A isoform, enolase 1 alpha non-neuron and glyceraldehyde-3-phosphate dehydrogenase (Table 2), as well as transcripts implicated in de novo fatty acid synthesis, such as ATP citrate lyase and fatty acid synthase, were upregulated by DHT (Table 2). Furthermore, two transcripts of the tri-acylglycerol synthesis pathway, namely, glycerol-3-phosphate dehydrogenase 1 (soluble) (GPD1) and diacylglycerol O-acyltransferase (DGAT) 1, were upregulated (Table 2). Genes involved in lipolysis, such as hormone-sensitive lipase, were also upregulated by DHT. Apolipoprotein E and low-density lipoprotein receptor-related protein 1 gene expression was increased by DHT, whereas a switch of transcript species of lipoprotein lipase (LPL) and monoglyceride lipase was observed (Table 2).

Transcripts implicated in energy metabolism (Table 3) as well as in amino-acid metabolism, nucleotide metabolism, transport metabolism, protein modification and general metabolism (Table 4), displayed numerous patterns of gene expression after androgen modulation. Cell division was also affected, since almost all the differentially expressed genes related to cell cycle, including cyclin I, and the genes associated with apoptosis, such as fat-specific gene 27, were upregulated by DHT (Table 5).

Some transcripts involved in RNA synthesis function displayed downregulation, except for the DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 5 and the CCAAT/enhancer binding protein (C/EBP) alpha transcripts, which were upregulated by DHT (Table 5). Genes associated with cell signaling, such as resistin and growth hormone receptor, were upregulated by DHT, whereas others, such as calmodulin 4, were downregulated (Table 5). Keratins (K), which are implicated in the cytoskeleton, were all downregulated by DHT (Table 6). On the other hand, other cytoskeletal components, such as actin beta cytoplasmic and gelsolin, as well as transcripts related to extra-cellular matrix, such as various procollagen isoforms, secreted acidic cysteine-rich glycoprotein (SPARC) and matrix metalloproteinase 2 (MMP-2), were upregulated by DHT (Table 6). Genes involved in cell and organism defense, such as superoxide dismutase 3 extracellular, glutathione peroxidases 3 and 4, and transcripts of the histocompatibility 2 complex, were upregulated by DHT (Table 7). In contrast, other transcripts, such as carbonic anhydrases, heat-shock proteins 1 and 4, adipsin (EST) and lysozyme, were down-regulated. Finally, transcripts associated with protein synthesis were affected in different ways by the androgen (Table 8). In addition, many novel transcripts were significantly differentially expressed (Table 9). Remarkably, 24 h after DHT injection, the tag CATG TTTGACAATGA was increased 353 times, whereas the tag CATG TCCCTATAAGC was decreased by almost 300-fold.

Among the 176 differentially expressed transcripts presented in Tables 2–8, excluding the transcripts from mitochondrial genome, 118 possessed one or more potential HRE in there promoter sequence. Moreover, 29 of these transcripts had one or more potential HRE 500 bp upstream of the transcription initiation start.

Discussion

There is much evidence that sex hormones mediate changes in adipose tissue distribution, the main observations indicating that there is a negative correlation between circulating testosterone levels and intra-abdominal fat mass and increased prognostic factors for atherosclerosis, risk of cardiovascular disease and diabetes (Seidell et al. 1990, Zumoff et al. 1990, Tsai et al. 2000). Through SAGE, we have characterized the effects of the nonaromatizable and most potent natural androgen, DHT, on the adipose tissue transcriptome. An overview of these data is presented by Fig. 1, which shows some of the changes mediated by the androgen on energy substrate pathways and adipocyte differentiation in male mice retro-peritoneal adipose tissue.

The general upregulation of transcripts involved in glycolysis may lead to a greater production of pyruvate which does not seem to be directed to the production of ATP, since lactate dehydrogenase 1 A chain is downregulated. Thus, fuel for de novo fatty acid synthesis may be produced, and transcripts involved in de novo fatty acid synthesis machinery itself are upregulated. In fact, positive regulation by DHT of lipogenic enzymes such as ATP citrate lyase, malic enzyme and fatty acid synthase has already been observed in monkey prostates (Arunakaran et al. 1992). In addition, two transcripts coding for lipoprotein lipase (LPL) are upregulated (except one EST), as previously reported (Anderson et al. 2002). It should be mentioned that even if there is fatty acid synthesis, if the fat is not stored in the form of triacylglycerol, and if fat oxidation is stimulated, fat mass may decrease. In fact, there is an upregulation by DHT of GPD1 and DGAT1, which are involved in triacyglycerol synthesis.

The upregulation of transcripts involved in de novo fatty acid and triacylglycerol synthesis could reflect the differentiation of preadipocytes into adipocytes, since many of these genes are stimulated in the adipocyte-differentiation process (Mackall et al. 1976, Coleman et al. 1978). In addition, C/EBP alpha, which is a major factor involved in adipocyte differentiation and in the expression of adipocyte-specific genes (Gregoire et al. 1998), is upregulated. Moreover, the present data also show an upregulation of DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 5 by DHT. The expression of this gene has been associated with adipogenesis (Kitamura et al. 2001), and it has been identified among the transcripts associated with adipose tissue fattening in the cow (Oishi et al. 2000). Cell structure reorganization is necessary for the differentiation process (Croissandeau et al. 2002). Keratin (K) proteins gather in pairs of acidic and basic keratins to form intermediate filaments. For example, in the skin, K5 and K14 form heterodimers, and alteration of one of these two molecules leads to skin fragility (Schuilenga-Hut et al. 2003). The downregulation of these and other keratins in this study may affect adipocyte cell shape. Extracellular matrix (ECM) components, such as collagen types I, III, IV, V and VI, increase three- to sixfold under adipogenic conditions (Nakajima et al. 2002). Moreover, MMP-2 is also involved in adipocyte development, since it regulates the balance between ECM deposition and degradation. In fact, inhibition of MMP can block the adipocyte differentiation process (Croissandeau et al. 2002). Transcripts related to all these ECM components are upregulated by DHT in the present study. All these mechanisms can promote the adipocyte differentiation process. It should be mentioned that the stimulation by DHT of collagen type 1 in osteoblastic cells (Kasperk et al. 1996) and of MMP-2 in human prostate cancer cells (Liao et al. 2003) has already been observed.

Besides lipogenesis, transcripts involved in the lipolysis process are also upregulated. In fact, hormone-sensitive lipase and carboxylesterase 3, a lipase participating in the mobilization of fatty acids from the triacylglycerol content of adipose tissue (Dolinsky et al. 2001), are upregulated by DHT, while monoglyceride lipase is also upregulated (except one EST). On the other hand, the adipocyte complement-related protein, also known as adiponectin, is upregulated. Knockout of this gene revealed an increased β-oxidation in muscle and liver of mice (Ma et al. 2002). Furthermore, knockout of the aP2 gene, encoding for adipocyte FABP4, has indicated a defect in basal and stimulated lipolysis (Coe et al. 1999). The present study shows that the expression of this gene is downregulated by DHT. Finally, fatty acid coenzyme A ligase long chain 2, which activates long-chain fatty acids for both lipid synthesis and degradation via β-oxidation (Weiner et al. 1991), is upregulated by DHT, the most potent androgen.

As presented in Tables 2–8, the changes in androgen state affected all cell functions at various levels. In our previous study on the adipose tissue transcritpome under intact animal condition, many genes involved in the cell and organism defense were among the most highly expressed (Bolduc 2004et al.). The present study shows that carbonic anhydrase 3, the most highly expressed gene in adipose tissue, is downregulated by DHT. It is still unclear why this gene is so highly expressed and what role it has in adipose tissue. In addition, the isoform carbonic anhydrase 5a is also downregulated. Generally, we have observed an upregulation by DHT of antioxidant proteins such as glutathione peroxidase 3 and 4, as well as superoxide dismutase 3 extracellular. Tags matching for the same gene or UniGene cluster were frequently found in the present study, particulary for the most abundant transcripts. For example, this was found for carbonic anhydrase 3. This may be partly explained by alternative polyadenylation cleavage site selection (Pauws et al. 2001) and alternative splicing (Mironov et al. 1999).

Analysis of all the data shows that various pathways are regulated by DHT in adipose tissue, and the gene expression profile changes induced by DHT suggest a promotion of fatty acid and triacylglycerol production as well as lypolysis in retroperitoneal adipose tissue. The equilibrium between these processes may bend on one side or the other, resulting in fat accumulation or fat loss. An in vitro study revealed that DHT could stimulate lipolysis through adenylate cyclase activation (Xu et al. 1990). However, it has been observed, in intact men, that DHT treatment increased visceral fat mass (Marin 1995). These findings on the acute effects of DHT seem to contradict the observations revealing a negative correlation between abdominal obesity and serum testosterone levels in men (Seidell et al. 1990, Zumoff et al. 1990, Tsai et al. 2000). In fact, testosterone treatment can reduce visceral fat mass and waist–hip ratio (WHR) in men (Marin et al. 1992, Marin 1995). Moreover, testosterone inhibited triacylglycerol uptake in abdominal adipose tissue of obese men (Marin et al. 1995). On the other hand, DHT had no significant effect on either WHR (Marin et al. 1995) or triacylglycerol uptake (Marin et al. 1995). Differential display PCR has already shown that testosterone and DHT have different effects on prostate gene expression (Avila et al. 1998). The different and sometimes opposite actions of testosterone and DHT may indicate that testosterone effects are mediated by a compound created via the aromatization process (Jensen 2000).

While DHT administration affected the expression of hundreds of genes, 7 days of gonadectomy affected only a few. In fact, only 13 classified transcripts were significantly differentially expressed between the intact and the GDX groups. Several of them, such as NADH dehydrogenase subunit 4, pheromone receptor V3R4 and three novel transcripts, showed an inverse pattern of expression in comparing the effect of castration and DHT injection. The tag CATG ATTTTCAGTTT, classified as a novel transcript, displayed a very sharp regulation by androgen modulation. The expression level of this tag changed from 7 in intact to 136 in GDX, falling back to 7 in DHT3h and rising to 115 in DHT24h. On the other hand, the expression of protamine 2, a transcript associated with chromatin condensation in sperm (Aoki & Carrell 2003), could never be restored after castration. This gene may be a target of testosterone, which may have different effects from DHT on gene expression.

Several HRE possibilities were found in the promoter of the significantly differentially expressed transcripts, many of them being included in the 500 bp upstream region of the transcription initiation start. Since the occurrence of a 6 bp sequence by chance alone is equal to once each 4096 pb, this finding reinforces the idea that these genes are potentially regulated by DHT.

In conclusion, the present data suggest that the administration of DHT to GDX male mice promotes processes involved in glycolysis, fatty acid and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation in retroperitoneal adipose tissue. Moreover, the steroid hormone affected almost all aspects of cell function by modulating hundreds of transcripts. In addition, many of those correspond to novel transcripts.

Table 1

General descriptive information about the four SAGE libraries analyzed

GDX+DHT
IntactGDX3 h24 hTotal
Tags sequenced45996455215165849256192431
Transcript species1766019768192072455361931
Well-characterized transcripts (%)3401 (19)3388 (17)4011 (21)4985 (20)8274 (13)
Partially characterized transcripts (%)1872 (11)1859 (10)2005 (11)2642 (11)5472 (9)
Novel transcripts (%)11783 (67)13887 (70)12542 (65)16006 (65)49798 (76)
Multiple matches (%)604 (3)634 (3)649 (3)920 (4)1387 (2)
Tags detected more than once433241275143500310136
Well-characterized transcripts (%)1685 (39)1580 (38)2252 (44)2301 (46)3919 (39)
Partially characterized transcripts (%)656 (15)600 (15)715 (14)732 (15)1545 (15)
Novel transcripts (%)1659 (38)1618 (39)1805 (35)1503 (30)3947 (39)
Multiple matches (%)332 (8)329 (8)371 (7)467 (9)725 (7)
Transcript species expressed .0.1%907210055144
Well-characterized transcripts (%)57 (63)43 (60)65 (65)33 (60)91 (63)
Partially characterized transcripts (%)10 (11)8 (11)10 (10)5 (9)14 (10)
Novel transcripts (%)10 (11)12 (17)9 (9)7 (13)20 (14)
Multiple matches (%)13 (15)9 (12)16 (16)10 (18)19 (13)
Table 2

Differentially expressed transcripts involved in sugar and lipid metabolism

TagsIG3 h24 hDescription (UniGene, Genbank)TGTTCT/AGAACA
I, intact; G, GDX; 3 h, DHT3h; 24 h, DHT24h.
*Significantly different (P<0.05) from G. Arrows are used to show a simplified representation of the effects of DHT. When castration induces a significant change in expression level, a dash is used instead of an arrow.
Sugar
CCTACTAACCA6148228*45↑ aldolase 1, A isoform (Mm. 16763, BC043026)-179/
CAAAAATAAAA02125*↑ enolase 1, alpha non-neuron (Mm. 90587, BC010685)/-1832,-948
GCCTCCAAGGA3722101*53↑ glyceraldehyde-3-phosphate dehydrogenase (Mm. 5289, AK081405)-1086/
TTGCTTTGTTG59647*↑ phosphoenolpyruvate carboxykinase 1, cytosolic (Mm. 42246, BC037629)/-607
GGACAGCACAC1973*37↑ pyruvate carboxylase (Mm. 1845, M97957)-1894,-1843,-504/
GCTTGTGACGA00017*↑ transaldolase 1 (Mm. 29182, BC04754)
GGATGCTGGGT24184894*↑ transketolase (Mm. 154387, AK012794)/-1325,-1249
TAAGGGAAATA30022*↑ triosephosphate isomerase (Mm. 4222, X53333)
CCAAATAAAAC12192*49↓ lactate dehydrogenase 1, A chain (Mm. 141443, U13687)
TTCCAGCTGCT5963239*↓ phosphoglycerate mutase 1 (Mm. 16783, BC002241)-1121,-469/-476
Lipid
TGCCTTCTCTG00012*↑ acylcoenzyme A dehydrogenase, very long chain (Mm. 18630, BC026559)-713/
GAACAGTCGAC15131299*↑ adipocyte complement-related protein (Mm. 3969, BC028770)-836/-865
CATCGCCAGTG11890398*118↑ apolipoprotein E (Mm. 156335, BC028816)/-382,-128
TTTGCTTTAAA811647*↑ ATP citrate lyase; RIKEN (Mm. 25316, BC021502; Mm. 45765, BC021413)-633,-391/-575
GAACATTTCAG10631*↑ EST ATP citrate lyase (Mm. 25316, BY687740)-633,-391/-575
TTGAGCTCTGA32122*↑ carboxylesterase 3 (Mm. 120807, BC019198)/-345
CTGCATAGCTC31119*↑ CD36 antigen (Mm. 18628, AK052825)/-926,-551,-51
TATGTCCACGA0115*12↑ diacylglycerol O-acyltransferase 1 (Mm. 22633, BC003717)
TGGGTGTCCAG12537*↑ fatty acid coenzyme A ligase, long chain 2 (Mm. 28962, AK004897)-1571,-429/-536
ATGCAGGGCCA4366520*126↑ fatty acid synthase (Mm. 3760, AF127033)
ACTCAATTCAG2226131*30↑ glycerol-3-phosphate dehydrogenase 1 (soluble) (Mm. 10669, BC019391)-156
GCTTCCTGAGC301016*↑ hormone-sensitive lipase (Mm. 1721, BC021642)/-516
GTCTAAAATTA83429*↑ lipoprotein lipase (Mm. 1514, AK002645)
ACAAGTCTCTG01137*↑ EST lipoprotein lipase (Mm. 1514, AA537700)
CAAAGCCCCAC12388*24↑ EST lipoprotein lipase (Mm. 1514, BE625478)
TGTAACAAATG20912*↑ long chain fatty acyl elongase (Mm. 26171, AK029029)
CTCAGTATCCC3219*6↑ low-density lipoprotein receptor-related protein 1 (Mm. 7221, X67469)
TTGTCAGGTAG414254*↑ malic enzyme, supernatant (Mm. 148155, J02652)-1545,-1277,-687/-1374
TGAGCATCGGG151675*41↑ monoglyceride lipase (Mm. 194795, AJ316580)-1068,-594/
GTCTGGGGGGA1679112427*↓ EST monoglyceride lipase (Mm. 194795, BE651758)-1068,-594/
GGCAAGTGCTA151935*↑ stearoyl-coenzyme A desaturase 1 (Mm. 140785, AF509570)
AAAACCATTGC29*8978358*— stearoyl-coenzyme A desaturase 1 (Mm. 140785, BC007474)
GCTGCCCTGGG1018*11↓ EST stearoyl-coenzyme A desaturase 1 (Mm. 140785, AA415297)
AGATAGATTTG12430*17↓ sterol carrier protein 2, liver (Mm. 1779, BC034613)-1000/-1479,-1136,-721,-656
TGGATGCCTTC3622211*↑ alcohol dehydrogenase 1, class I (Mm. 2409, BC013477)-1001,-295/-1938,-1895
GACACCAGAGC41210*↓ brain acyl-CoA hydrolase (Mm. 197523, BC013507)/-1466,-1012
AAGACCTATGT51926595*65*↓ diazepam binding inhibitor (Mm. 2785, BC028874)-1234/
AGCCAAAGGAA27638073*273↓ fatty acid binding protein 4, adipocyte (Mm. 582, BC002148)-415/-1542,-1492
Table 3

Differentially expressed transcripts involved in energy metabolism

TagsIG3 h24 hDescription (UniGene, Genbank)TGTTCT/AGAACA
I, intact; G, GDX; 3 h, DHT3h; 24 h, DHT24h.
*Significantly different (P<0.05) from G. Arrows are used to show a simplified representation of the effects of DHT. When castration induces a significant change in expression level, a dash is used instead of an arrow.
**Tags matching the mitochondrial genome (Genbank accession no. NC_001569); values listed for these tags indicate the locus within the mitochondrial genome.
CAACTGTATTT11220*↑ aconitase 2, mitochondrial (Mm. 154581, BC004645)-390/
CATCTTCAGCC201349*16↑ ATPase, Ca++ transporting, cardiac muscle, fast twitch 1(Mm. 35134, AY081946)
CGGGAGATGCT21116*↑ ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit (Mm. 41, BC012241)
CAGGCCACACA15045*ATP synthase, H+ transporting mitochondrial F1 complex, β subunit (Mm. 103838, AK010314)
ATAATACATAA190139102346*ATP synthase F0 subunit 6 (8593–8607)**
TAGATATAGGC9911628*38*ATP synthase F0 subunit 6 (8596–8582)
TTGATGTATCT00017*ATP synthase F0 subunit 8 (7791–7777)
AGGACAAATAT2536224*138*cytochrome b (14539–14553)
AATATGTGTGG53129*↑ cytochrome c oxidase, subunit VIc (Mm. 548, BC024666)
TATTGGCTCTG00136*↑ cytochrome c oxidase, subunit VIIIa (Mm. 14022, AK002218)-1524/-703,-641
AGGAGGACTTA271748*NADH dehydrogenase subunit 2 (4410–4424)
GCTGCCCTCCA732568131*330cytochrome c oxidase subunit 1 (6813–6827)
TGGTGTAAGCA939812*4*cytochrome c oxidase subunit 1 (6676–6662)
ATGAGAACAGC16251*1*cytochrome c oxidase subunit 1 (6731–6717)
AAGTCATTCTA23681*7*cytochrome c oxidase subunit 1 (6816–6802)
GGCAGTTACGA1745*17cytochrome c oxidase subunit 1 (5511–5497)
TAGTTACTTAC91913127*cytochrome c oxidase subunit 1 (6093–6107); NEW1 domain containing protein (Mm. 22338, AK046719)
AGCAGTCCCCT62555033682*cytochrome c oxidase subunit 2 (7497–7511)
AGTGGAGGACG132914026*cytochrome c oxidase subunit 2 (7500–7486)
CTGCGGCTTCA42416*5*cytochrome c oxidase subunit 3 (9325–9311)
AGCAATTCAAA7242*27NADH dehydrogenase subunit 3 (9682–9696)
GTAGTGGAAGT451109*26*NADH dehydrogenase subunit 3 (9685–9671)
ATGACTGATAG259*696179*162*NADH dehydrogenase subunit 4 (11230–11244)
GAGTTTGGATT122554*NADH dehydrogenase subunit 4 (11080–11066)
ATTATAGTACG91930*NADH dehydrogenase subunit 4 (11192–11178)
GTTTTGGATTA221544*NADH dehydrogenase subunit 4 (10671–10657)
Table 4

Differentially expressed transcripts involved in other metabolism

TagsIG3 h24 hDescription (UniGene, Genbank)TGTTCT/AGAACA
I, intact; G, GDX; 3 h, DHT3h; 24 h, DHT24h.
*Significantly different (P<0.05) from G. Arrows are used to show a simplified representation of the effects of DHT. When castration induces a significant change in expression level, a dash is used instead of an arrow.
Amino acid
GAAACTCTACT732136*↑ cysteine dioxygenase 1, cytosolic (Mm. 29996, BC013638)-1838/-1884
TATAGTATGTT10035*↑ glutamate-ammonia ligase (glutamine synthase) (Mm. 2338, AY044241)
Nucleotide
TCCTTGGGGGT18219*7↓ histidine triad nucleotide binding protein (Mm. 425, AK012433)-1300,-358/-1311,-384
GTGCTGCCAGT17458*1*↑ ectonucleotide pyrophosphatase/phosphodiesterase 2 (Mm. 28107, AF123542)/-1508
Transport
GTCAATGACGT3015*7↑ aquaporin 1 (Mm. 18265, BC007125)
TCAGGCTGCCT1511472*↑ ferritin heavy chain (Mm. 1776, BC012314)-1572,-599/-1289,-546
CCCTGGGTTCT1024149*58↑ ferritin light chain 1; ESTs similar to ferritin L subunit 2; EST RIKEN (Mm. 7500, BC019840; Mm. 220829, BQ950380; Mm. 34374, BI789635)
CTTCTCATTTG21318*↑ lyosomal-associated protein transmembrane 4A (Mm. 30071, AK084515)-1464/-1610
AATTTCTTCCT00093*↑ major urinary protein 1 and 2 (Mm. 157893, BC012221; Mm. 4516, BC012259)
CAGAAGAAGCT00029*↑ EST major urinary protein 1 (Mm. 157893, CA457333)
AGTCTCGAGGG15138*↑ solute carrier family 1, member 7 (Mm. 1056, BC037462)/-1252
GTCAGGTCACA3115*16*↑ solute carrier family 25 (mitochondrial carrirer; citrate transporter), member 1 (Mm. 229291, BC037087)
Protein modification
CAGGTGTCCAC14*031— EST protein tyrosine phosphatase 4a2; EST RIKEN (Mm. 193688, AV049645; Mm. 36280, AU024264)-836/
General
TGGAGATAAGC2326*23*↑ acid phosphatase 5, tartrate resistant (Mm. 46354, BC019160)/-204
Table 5

Differentially expressed transcripts involved in cell division, RNA synthesis and cell signalling

TagsIG3 h24 hDescription (UniGene, Genbank)TGTTCT/AGAACA
I, intact; G, GDX; 3 h, DHT3h; 24 h, DHT24h.
*Significantly different (P<0.05) from G. Arrows are used to show a simplified representation of the effects of DHT. When castration induces a significant change in expression level, a dash is used instead of an arrow.
Cell cycle
TACTGCTGATA00013*↑ cyclin I (Mm. 22711, BC003290)/-507
CTGTTTCAAGG12434*12↑ G0/G1 switch gene 2 (Mm. 3283, AK003165)-645/-1831,-595
GCGGCGGATGG8840257*72↑ lectin, galactose binding, soluble 1 (Mm. 43831, NM_008495)
Apoptosis
GCTTATAGATC2219*1↑ B-cell receptor-associated protein 31 (Mm. 17, BC002106)
CAGCTGCCTCT40376*↑ fat specific gene 27 (Mm. 10026, AK080133)-575/-300
TGGGTTGTCTA346012*76↓ tumor protein, translationally-controlled 1 (Mm. 254, X06407)/-595
Chromosome structure
TCGATGTCTGA23*000— protamine 2 (Mm. 541, AK005729)
Meiosis
TCAACAAGCAC3150*10↓ EST synaptonemal complex protein 3 (Mm. 148209, AW490250)
RNA processing
GCCTTCCAATA10112*↑ DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 5 (Mm. 19101, BC009142)/-170
GGCAGCACAAA151290*↓ EST heterogeneous nuclear ribonucleoprotein L (Mm. 9043, BB399813)-1466/
Transcription factors
CTAGATGTCGT01117*↑ C/EBP3 (Mm. 34537, BC011118)
ACGCAGTGGGT91470*↓ EST nuclear factor I/A (Mm. 4771, AK004196)/-1885,-1849
General
CACGTGCCTGA3821101*↓ zinc-finger homeobox 1b (Mm. 37676, AF033116)/-852
Cell adhesion
TGAGCTTTGGG9026*8↑ melanoma cell adhesion molecule (Mm. 39103, BC026985)/-1377,-1151
CAATGTGGGTT00020*↑ osteoblast specific factor 2 (fasciclin I-like) (Mm. 10681, BC031449)
TGCAGGTGCAC2221193*↓ EST integrin 3 1 (fibronectin receptor 3) (Mm. 4712, BB109045)/-634,-39
Effector modulator
ACTCGGAGCCA6042217*↓ calmodulin 1 (Mm. 34246, AK004673)-1928/-1360,-965
AGTGAGGAAGA14150*1*↓ calmodulin 4 (Mm. 21075, AK009664)/-1901,-829
TTTTTGAACAA2634133*↓ catenin beta (Mm. 3476, BC006739)
TGCACACAACT24291*0*↓ S100 Ca binding protein A3 (Mm. 703, AF004941)-1160/-1518,-668,-525
TATCCCACGCC13024*↑ S100 Ca binding protein A11 (calizzarin) (Mm. 175848, BC021916)-1504,-872/-1869
Hormone/growth factors
CCACTGTGTCC9770*26↑ resistin (Mm. 1181, AF290870)-1877/
Receptors
CATACGCATAA30018*↑ growth hormone receptor (Mm. 3986, BC024375)-411,-366,-292,-231,-222/-1064
GTCTGCTGATG77*235323— guanine nucleotide binding protein, beta 2, related sequence 1(Mm. 5305, X75313)
GAATATGCAGC4190*0*↓ kinase insert domain protein receptor (Mm. 285, AK031739)/-1554
CAAACTTTATA8*375*2*— pheromone receptor V3R4 (Mm. 160379, AF324869)-1599,-1094/
Table 6

Differentially expressed transcripts involved in cytoskeletal and extracellular matrix

TagsIG3 h24 hDescription (UniGene, Genbank)TGTTCT/AGAACA
I, intact; G, GDX; 3 h, DHT3h; 24 h, DHT24h.
*Significantly different (P<0.05) from G. Arrows are used to show a simplified representation of the effects of DHT. When castration induces a significant change in expression level, a dash is used instead of an arrow.
Cytoskeletal
CCCTGAGTCCA3930117*33↑ actin, beta, cytoplasmic (Mm. 297, X03672)-778/-1950,-1350,-1078
GATACTTGGAA142092*68*↑ EST actin, beta, cytoplasmic (Mm. 297, BY464177)-778/-1950,-1350,-1078
CTCCTGGACAC1526258*128*↑ gelsolin (Mm. 21109, NM_146120)-922,-857,-848/-395
GAGCAGACCGT382589*19↑ myosin, heavy polypeptide 4, skeletal muscle (Mm. 35531, AJ278733)/-1045
GTGATTGCTAAG204*497421— EST myosin light chain, phosphorylatable, fast skeletal muscle
(Mm. 252182, AV082184; Mm. 249289, AV214319; Mm. 14526, AK010483; Mm. 251434, AV148670)-1509/-857,-124
ACCTCTCAGAT0013*9*↑ pericentrin 2; ferritin light chain 1; EST ferritin light chain 2 (Mm. 4379, U05823; Mm. 7500, AK002547; Mm. 30357, NM_008049)
GGCTGGGGGCT7938*11↑ profilin 1 (Mm. 2647, BC002080)
TTGGTGAAGGA110733*↑ thymosin, beta 4, X chromosome (Mm. 142729, BC018286)-1449,-657/-291
ATGTCTCAAAG75144*↑ EST tubulin, alpha 1; 2; 6 (Mm. 196396, BC008117; Mm. 197515, AK075955; Mm. 88212, BB001495)
CTGCTCAGGCT41471*0*↓ keratin complex 1, acidic, gene 14 (Mm. 6974, BC011074)-1160/-1296
CCCAGAGCACT14181*0*↓ keratin complex 2, basic, gene 1 (Mm. 18137, AK019521)-1937/
TTCTTTGGTGA39486*0*↓ keratin complex 2, basic, gene 5 (Mm. 22657, BC006780)
TGGTGCACTTC25320*0*↓ keratin complex 2, basic, gene 6g (Mm. 89769, AB033744)
GAGGGCCGGAA141596613*↓ troponin I, skeletal, fast 2 (Mm. 39469, BC028515)/-1022
Extracellular matrix
GCATTGAAAG04027*↑ dermatopontin (Mm. 28935, AF143374)-1726/-526,-399
TGCCGGATGAC21*2134— dermatopontin (Mm. 28935, AK019890)-1726/-526,-399
GGAAATGGCAA00014*↑ matrix metalloproteinase 2 (Mm. 29564, M84324)-1957,-965,-884,-767/
GTTCCAAAGAA10014*↑ EST procollagen, type I, alpha 2 (Mm. 4482, AK075707)-1157/
TCTTCTATGCA38493311*↓ EST procollagen, type I, alpha 2 (Mm. 4482, BQ126567)-1157/
TGTTCATCTTG54374*↑ procollagen, type III, alpha 1 (Mm. 147387, AK041115)-708/
GTGTCTGATAA3836*10↑ procollagen, type IV, alpha 1 (Mm. 738, J04694)-1586,-1058/
GTGCTGCCCTG2119*3↑ procollagen, type V, alpha 3 (Mm. 30477, AF176645)/-400
GCTCCCCCACA10017*↑ procollagen, type VI, alpha 1 (Mm. 2509, X66405)-955/-1538,-1162
CAAACTCTCAC22205100*↑ secreted acidic cysteine rich glycoprotein (Mm. 35439, AK014286)/-424
GAACATTGCAC3245135*61↑ secreted acidic cysteine rich glycoprotein (Mm. 35439, BC004638)/-424
General
TAAGTAGCAAA11148*↑ integral membrane protein 2B (Mm. 4266, BC021786)-1449/
TTTCCTTCAAC5049176*↓ EST clathrin, light polypeptide (Lca) (Mm. 198817, B1736877)-75/
GGGTTGGCCCA141850*↓ nidogen 2 (Mm. 20348, AB017202)
Table 7

Differentially expressed transcripts involved in general homeostasis, stress response and immunity

TagsIG3 h24 hDescription (UniGene, Genbank)TGTTCT/AGAACA
I, intact; G, GDX; 3 h, DHT3h; 24 h, DHT24h.
*Significantly different (P<0.05) from G. Arrows are used to show a simplified representation of the effects of DHT. When castration induces a significant change in expression level, a dash is used instead of an arrow.
General
CCCTGCCTTAA02419*↑ creatine kinase, muscle (Mm. 2375, AK009950)/-745
GAAAAGTGGAT2016*6↑ epoxide hydrolase 2, cytoplasmic (Mm. 15295, BC015087)-936/
CTATCCTCTCA22191293*↑ glutathione peroxidase 3 (Mm. 7156, AK002219)-229/-1647
AAGGTCTGCCT131998*33↑ glutathione peroxidase 4 (Mm. 2400, D87896)
GAAGAGGGGGA74*152871*— haptoglobin (Mm. 26730, M96827)-1792,-1191/-820,-545
GGGGGAGTGGA2024*4↑ neuronatin (Mm. 140956, BC036984)/-151
TTTCCAGGTGT00224*↑ selenoprotein W, muscle 1 (Mm. 42829, NM_009156)
TATCTGTGCAT296514*13*↓ selnoprotein P, plasma, 1 (Mm. 22699, X99807)/-556
TTCCCGATCAC8434*10↑ superoxide dismutase 3, extracellular (Mm. 2407, BC010975)/-1267
CTACGTTCTCT10012*↑ thioredoxin-like 2 (Mm. 29675, AK010354)-846,-313/-1003
CCCTGAGGGGT8951279*79↑ transferrin (Mm. 37214, BC012313)-1703,-673/-1061
AGCAAGATGGT214612*2*↓ aminolevulinic acid synthase 1 (Mm. 19143, BC022110)-1396/
CCTATTAAAAA9871185813239*↓ carbonic anhydrase 3 (Mm. 300, BC011129)
AATTTCACACC90*232142451— carbonic anhydrase 3 (Mm. 300, M27796)
GGTGTGTTTTA28532315*↓ EST carbonic anhydrase 3 (Mm. 300, AV291195)
GGAGGCAGAGG1016101*↓ carbonic anhydrase 5a, mitochondrial (Mm. 116761, BC030174)/-1582,-476
TGAACCGTCCC33274*8↓ glutathione S-transferase, pi 2 (Mm. 426, BC002048)-1262,-1185/
Stress response
TATTAGTCTTA3027135*↓ heat-shock protein, 1 (Mm. 1843, AK004658)/-629
CTGAGCAGAAT10254*1*↓ heat-shock protein 4 (Mm. 1032, BC003770)-1383,-996/-1768,-1341
GAATAATAAAA31218*↑ heat-shock protein 8; EST heat shock cognate hsc 73; ESTs (Mm. 197551, BC006722; Mm. 258783, BY761643; Mm. 247242, BY415840)
Immunity
GAGTGGATTCT10014*↑ Cd63 antigen (Mm. 4426, BC012212)
GTTGTTTTCCA20012*↑ Fc receptor, IgG, alpha chain transporter (Mm. 3303, BC003786)
GATTGAGAATG1114555*↑ EST histocompatibility 2, D region locus I; L region; histocompatibility 2, K region (Mm. 33263, AW 741231; Mm. 196214, BY576457; Mm. 16771, BC011306)
TCACACATTGC0014*4↑ histocompatibility 2, Q region locus 10 (Mm. 88795, K00614)/-627
GTTCAAGTGAC55150*↑ Ia-associated invariant chain (Mm. 258212, BC003476)-1765/
CTAATATTTGC00012*↑ immunoglobulin kappa chain, variable 8; 28; constant region; EST RIKEN (Mm. 104747, BC028925; Mm. 220176, BC021781; Mm. 222734, X02816; Mm. 255225, AK008450)
GAGGACTGCCA281657*14↑ lymphocyte antigen 6 complex, locus E (Mm. 788, BC019113)
CATCTGAAAAA21541175*704↓ EST adipsin (Mm. 4407, AW215391)
TGTCAGTCTGT122839226*↓ lysozyme (Mm. 45436, BC002069)-581/
Table 8

Differentially expressed transcripts involved in protein synthesis

TagsIG3 h24 hDescription (UniGene, Genbank)TGTTCT/AGAACA
I, intact; G, GDX; 3 h, DHT3h; 24 h, DHT24h.
*Significantly different (P<0.05) from G. Arrows are used to show a simplified representation of the effects of DHT.
Post-translational modification/targeting
TGAACACTGAA11118*↑ transglutaminase 2, C polypeptide (Mm. 18843, BC016492)-1456,-199/
TAGCTTCCTCT00112*↑ sequestosome 1; EST RIKEN (Mm. 200125, BC006019; Mm. 41784, BM207023)
Protein turnover
CAGATCTTTGT2927110*43↑ ubiquitin C; EST RIKEN (Mm. 331, BC021837; Mm. 41423, BB476893)-513/
GTAAGCATAAA12019*↑ EST ubiquitin B (Mm. 235, BU529368)
TGACCCCGGGA12124*↑ ubiquitin A-52 residue ribosomal protein fusion product 1 (Mm. 43005, BC014772)
GTGGAGGCGCC99882*3*↓ cystatin E/M (Mm. 36816, NM_028623)-782/-1417,-1261
GAGTAAGGACA9120*1↓ kallikrein 7 (chymotryptic, stratum coneum) (Mm. 34974, BC027823)/-561
Translation factors
GAGCTCCAGCG18335*4↓ eukaryotic translation initiation factor 4E binding protein 1(Mm. 6700, BC002045)-1790,-846/-1808,-920,-816
TGCAATATGGC223072*↓ EST eukaryotic translation initiation factor 4A2 (Mm. 16323, BY674584)
Ribosomal proteins
GGATTTGGCTT118244*↑ ribosomal protein, large P2 (Mm. 14245, BC012413)-859/-463
AACAATTTGGG31037*↑ ribosomal protein L9 (Mm. 14244, BC013165)
TGGTCAGGATC171220*↓ EST ribosomal protein L9 (Mm. 14244, AV290443)
ACATCATAGAT00016*↑ ribosomal protein L12 (Mm. 70127, BC018321)-1781/-1911
TGGATCAGTCT32250*↑ ribosomal protein L19 (Mm. 30806, BC010710)-1598,-374/-1653
CCAGAACAGAC85132*↑ ribosomal protein L30 (Mm. 3487, BC002060)-1200,-93/-204
GTGAAACTAAA54232*↑ ribosomal protein S4, X-linked (Mm. 66, BC009100)-1468,-380/-1558
GACCTGGAGCC331383*24↑ EST ribosomal protein S14 (Mm. 43778, AV094184)-1809,-221/
AATTTCAAAAC31028*↑ ribosomal protein S17 (Mm. 42767, BC002044)/-862
CTGTAGGTGAT00123*↑ ribosomal protein S23 (Mm. 30011, BC002145)
TAAAGAGGCCG30028*↑ ribosomal protein S26 (Mm. 372, BC036987)/-374
GGCTTCGGTCT163145*↓ ribosomal protein, large, P1; EST RIKEN (Mm. 3158, AK010656; Mm. 233844, AI529467)-1798/
GTTGCTGAGAA2271457529*↓ ribosomal protein 10 (Mm. 100113, BC024901)/-939,-660
GAGGAGAAGAA17214532*18*↓ ribosomal protein L3 (Mm. 3486, BC009655)-1476/-1678.-1352,-1158
CTGCTATCCGA1411126110*↓ ribosomal protein L5 (Mm. 4419, BC026934)-1900,-1022/
AATCCTGTGGA6844469*↓ ribosomal protein L8 (Mm. 30066, U67771)-1256,-704,-628/
CCCACAAGGTA86673014*↓ ribosomal protein L27 (Mm. 28985, BC024366)/-244
ATCCGAAAGAA2845409*↓ ribosomal protein L28 (Mm. 3111, BC024395)
AAAACAGTGGC80756820*↓ ribosomal protein L37a (Mm. 21529, NM_009084)/-85
GGGAAGGCGGC170885823*↓ ribosomal protein S3a (Mm. 6957, BC039659)-1876/
TATGTCAAGCT2111008425*↓ ribosomal protein S12 (Mm. 21289, BC018362)
CCTACCAAGAC1711186828*↓ ribosomal protein S20 (Mm. 21938, BC011323)/-1469,-1419,-1160
GCCTTTATGAG1431055823*↓ ribosomal protein S24 (Mm. 16775, AK002986)-589/
GATGACACCAG138668113*↓ ribosomal protein S28 (Mm. 200920, BC010987)/-1690,-1556,-1375
CTAGTCTTTGT13222*42↓ ribosomal protein S29 (Mm. 154915, BC024393)-1560/
TTCAGCCCGTA1316311*↓ EST ribosomal protein S29 (Mm. 154915, CA787284)-1560/
Table 9

Novel transcripts differentially expressed

TagsIG3 h24 h
I, intact; G, GDX; 3 h, DHT3h; 24 h, DHT24h.
*Significantly different (P<0.05) from G. Arrows are used to show a simplified representation of the effects of DHT. When castration induces a significant change in expression level, a dash is used instead of an arrow.
ATTTTCAGTTT7*1367*115
TCCTACAGTGG2*202*7
GAAAACGAGAA0*1325
TTTGACAATGA0185*353*
TTATAGACGGC11158*74*
GTCACCTTTCG0031*47*
GGCTGCGGCCT37288*166*
CTTCCCCGGGA0747*26
GCCTCCTGGGT2040*18*
GATGGAGTGAC1125*3
GGTGACCACAC1412125*28
CTGACGACTGA9959*16
TTGAGTCCTCC2031*10
ATGGGTCAAAG0227*11
TCGGCTCCGAG2127*8
ACTGGGCAGGA0015*9
TTCCAAAGCAA011383*
GCGGAGATGAG5022*14*
GGCGGGACCAC16444*7
GATGCGCTTGT13425*3
GCAGCCAGGGC4223*3
GTGGCGGTGGC1014*2
TCCGGAGAAAA00142*
TCCGGAGAAAG00549*
GCCTGAATCAG11982*
GGACAATTGTG00219*
TAGCTGTGTGG00115*
CAAGTAGATGA00115*
CAGTCAGAAAG00014*
CAGCTAGTTGC00122*
AAGGAATAAGC271231*
GGCTAGATTTC321124*
TGTGATGTCAG00214*
TAATCATCGAA831328*
TGGGAGATGCT20915*
CCCTATTAAGC555415*0*
CATCATAAAAA437337123*5*
GCAGTGGGTAG23850095*55*
TCACCGTACAT22223*0*
GCGGAGAAGAA21190*0*
CCCTTTCATAA10710418*16*
CAATGCTGCCT32202*3
TAGAGACTGCC23582*14*
AAAAATCATCG41494*48
GTGACCACGGG61409*30
TCCCTATAAGC199298115*0*
TGTCAGGTGTC184310*0*
TCCCCGACATC17488*1*
TCTCCGTACAT366312*6*
TCCCTGTTAAG12182*0*
ATCTCACCCAG21496*10*
TCCACGTACAT9182*0*
GTGCAGGGGTG15234*2*
GCTCTAGCTGC4180*1*
AGTCAGATTTC7140*0*
ACAGCAAGGGT5140*0*
GATGGAGACGG5223*3*
ATAGACTTTCA8120*0*
ATCTCGAGAGG5151*2
TCCCCGTACAA3642160*
TCCCCGTACAC6278260*
TCCCCGTACAG4052262*
CCGATGATCAG3730342*
CCCCTATTAAG3025211*
CATCATAAAAC3330221*
CATCATAAAAG3930172*
TCCTATTAAGC5390410*
TCCCTATTAAA3937231*
TCCCTATTAAC2931211*
TCCCTATTAAT2824170*
CAACCATCATC9951806*
CAAAGATTAAA1962058751*
ATGTTGGGCAG281671*
GAAGCACACAG232360*
TCCCCATACAT122030*
TCCCCTATTAA4743247*
CATCATAAAAT3232101*
GCCCTATTAAG151990*
TCCATATTAAG162251*
TCCGCGTACAT152051*
TCCCTATTAGG221551*
TACCCGTACAT131440*
TTCTGGTTTGT55712710*
TCACTATTAAG1119130*
TACCTATTAAG101990*
TCCCCTACATC91850*
TCCGTATTAAG1826113*
TGCCTATTAAG1623112*
TCCCATTAAGC102391*
TCCCTACTAAG142572*
CTTAACTCTGC1525102*
TCCCCCGTACA122151*
TCGCCGTACAT121951*
TTCCTATTAAG1623123*
TCCCGTACATC101320*
TCCCTTTAAGC41320*
CATCATACATC212243*
TCCCTATAAGA81260*
TCGCTATTAAG1519112*
CCTATTAAAAG1419112*
Figure 1
Figure 1

Overview of the effects of dihydrotestosterone on energy substrate pathways and adipocyte differentiation in retroperitoneal adipose tissue. Abbreviations: ACADVL, acyl-coenzyme A dehydrogenase very long chain; ACLY, ATP citrate lyase; ACTβ, actin beta cytoplasmic; ALDO1, aldolase 1 A isoform; APOE, apolipoprotein E; CCNI, cyclin I; C/EBPα, CCAAT/enhancer binding protein alpha; CES3, carboxylesterase 3; chol, cholesterol; COL1α2,3α1,4α1,5α3,6α1, procollagen type I alpha 2, type III alpha 1, type IV alpha 1, type V alpha 3, type VI alpha 1; CPT2, carnitine palmitoyltransferase 2; DDX5, DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 5; DGAT1, diacylglycerol O-acyltransferase 1; ENO1, enolase 1 alpha nonneuron; ECM, extracellular matrix; FA, fatty acids; FABP4, adipocyte fatty acid-binding protein 4; FAE, fatty acyl elongase; FAS, fatty acid synthase; G0S2, G0/G1 switch gene 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHR, growth hormone receptor; GPD1, glycerol-3-phosphate dehydrogenase 1 (soluble); GSN, gelsolin; HSL, hormone-sensitive lipase; KRT1,5,6 g,14, keratin genes 1, 5, 6 g and 14; LDH1, lactate dehydrogenase 1 A chain; LPL, lipoprotein lipase; LRP1, low-density lipoprotein receptor-related protein 1; ME, malic enzyme supernatant; MGL, monoglyceride lipase; MYH4, myosin heavy polypeptide 4 skeletal muscle; MMP2, matrix metalloproteinase 2; nascent-lp, nascent lipoprotein; PCX, pyruvate carboxylase; PGAM1, phosphoglycerate mutase 1; SCD1, stearoyl-coenzyme A desaturase 1; SLC25, solute carrier family 25 (mitochondrial carrier; citrate transporter) member 1; SPARC, secreted acidic cysteine-rich glycoprotein; TALDO1, transaldolase 1; TG, triglyceride; TKT, transketolase; TPI, triosephosphate isomerase; TUBα1,2,6, EST tubulin alpha 1, 2 and 6; VLDL, very low-density lipoprotein. *Except one EST.

Citation: Journal of Molecular Endocrinology 33, 2; 10.1677/jme.1.01503

We would like to thank Nicolas Lafond, Dr André Boivin, and all the research assistants involved in the ATLAS project for their skillful technical assistance. This work was supported by Genome Québec and Genome Canada.

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  • KitamuraA Nishizuka M Tominaga K Tsuchiya T Nishihara T & Imagawa M 2001 Expression of p68 RNA helicase is closely related to the early stage of adipocyte differentiation of mouse 3T3-L1 cells. Biochemical and Biophysical Research Communications287435–439.

    • Search Google Scholar
    • Export Citation
  • LashAE Tolstoshev CM Wagner L Schuler GD Strausberg RL Riggins GJ & Altschul SF 2000 SAGEmap: a public gene expression resource. Genome Research101051–1060.

    • Search Google Scholar
    • Export Citation
  • LiaoX Thrasher JB Pelling J Holzbeierlein J Sang QX & Li B 2003 Androgen stimulates matrix metalloproteinase-2 expression in human prostate cancer. Endocrinology1441656–1663.

    • Search Google Scholar
    • Export Citation
  • MaK Cabrero A Saha PK Kojima H Li L Chang BH Paul A & Chan L 2002 Increased beta-oxidation but no insulin resistance or glucose intolerance in mice lacking adiponectin. Journal of Biological Chemistry27734658–34661.

    • Search Google Scholar
    • Export Citation
  • MackallJC Student AK Polakis SE & Lane MD 1976 Induction of lipogenesis during differentiation in a ‘preadipocyte’ cell line. Journal of Biological Chemistry2516462–6464.

    • Search Google Scholar
    • Export Citation
  • MarinP1995 Testosterone and regional fat distribution. Obesity Research3 (Suppl 4) 609S–612S.

  • MarinP Krotkiewski M & Bjorntorp P 1992 Androgen treatment of middle-aged obese men: effects on metabolism muscle and adipose tissues. European Journal of Medicine1329–336.

    • Search Google Scholar
    • Export Citation
  • MarinP Oden B & Bjorntorp P 1995 Assimilation and mobilization of triglycerides in subcutaneous abdominal and femoral adipose tissue in vivo in men: effects of androgens. Journal of Clinical Endocrinology and Metabolism80239–243.

    • Search Google Scholar
    • Export Citation
  • MironovAA Fickett JW & Gelfand MS 1999 Frequent alternative splicing of human genes. Genome Research91288–1293.

  • NakajimaI Muroya S Tanabe R & Chikuni K 2002 Extracellular matrix development during differentiation into adipocytes with a unique increase in types V and VI collagen. Biology of the Cell94197–203.

    • Search Google Scholar
    • Export Citation
  • NelsonCC Hendy SC Shukin RJ Cheng H Bruchovsky N Koop BF & Rennie PS 1999 Determinants of DNA sequence specificity of the androgen progesterone and glucocorticoid receptors: evidence for differential steroid receptor response elements. Molecular Endocrinology132090–2107.

    • Search Google Scholar
    • Export Citation
  • NovakJP Sladek R & Hudson TJ 2002 Characterization of variability in large-scale gene expression data: implications for study design. Genomics79104–113.

    • Search Google Scholar
    • Export Citation
  • OishiM Taniguchi Y Nishimura K Yamada T & Sasaki Y 2000 Characterisation of gene expression in bovine adipose tissue before and after fattening. Animal Genetics31166–170.

    • Search Google Scholar
    • Export Citation
  • PauwsE van Kampen AH van de Graaf SA de Vijlder JJ & Ris-Stalpers C 2001 Heterogeneity in polyadenylation cleavage sites in mammalian mRNA sequences: implications for SAGE analysis. Nucleic Acids Research291690–1694.

    • Search Google Scholar
    • Export Citation
  • SchenaM Shalon D Davis RW & Brown PO 1995 Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science270467–470.

    • Search Google Scholar
    • Export Citation
  • Schuilenga-HutPH Vlies P Jonkman MF Waanders E Buys CH & Scheffer H 2003 Mutation analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa simplex and identification of novel mutations. Human Mutations21447.

    • Search Google Scholar
    • Export Citation
  • SeidellJC2000 Obesity insulin resistance and diabetes – a worldwide epidemic. British Journal of Nutrition83 (Suppl 1) S5–8.

  • SeidellJC Bjorntorp P Sjostrom L Kvist H & Sannerstedt R 1990 Visceral fat accumulation in men is positively associated with insulin glucose and C-peptide levels but negatively with testosterone levels. Metabolism39897–901.

    • Search Google Scholar
    • Export Citation
  • SheehanMT & Jensen MD 2000 Metabolic complications of obesity. Pathophysiologic considerations. Medical Clinics of North America84363–385 vi.

    • Search Google Scholar
    • Export Citation
  • SjostromL Smith U Krotkiewski M & Bjorntorp P 1972 Cellularity in different regions of adipose tissue in young men and women. Metabolism211143–1153.

    • Search Google Scholar
    • Export Citation
  • St-AmandJ Okamura K Matsumoto K Shimizu S & Sogawa Y 2001 Characterization of control and immobilized skeletal muscle: an overview from genetic engineering. FASEB Journal15684–692.

    • Search Google Scholar
    • Export Citation
  • TsaiEC Boyko EJ Leonetti DL & Fujimoto WY 2000 Low serum testosterone level as a predictor of increased visceral fat in Japanese-American men. International Journal of Obesity-Related Metabolic Disorders24485–491.

    • Search Google Scholar
    • Export Citation
  • VagueJ1956 The degree of masculine differentiation of obesities: a factor determining predisposition to diabetes atherosclerosis gout and uric calculous disease. American Journal of Clinical Nutrition420–34.

    • Search Google Scholar
    • Export Citation
  • VelculescuVE Zhang L Vogelstein B & Kinzler KW 1995 Serial analysis of gene expression. Science270484–487.

  • VelculescuVE Zhang L Zhou W Vogelstein J Basrai MA Bassett DE Jr Hieter P Vogelstein B & Kinzler KW 1997 Characterization of the yeast transcriptome. Cell88243–251.

    • Search Google Scholar
    • Export Citation
  • VirlonB Cheval L Buhler JM Billon E Doucet A & Elalouf JM 1999 Serial microanalysis of renal transcriptomes. PNAS9615286–15291.

  • WeinerFR Smith PJ Wertheimer S & Rubin CS 1991 Regulation of gene expression by insulin and tumor necrosis factor alpha in 3T3-L1 cells. Modulation of the transcription of genes encoding acyl-CoA synthetase and stearoyl-CoA desaturase-1. Journal of Biological Chemistry26623525–23528.

    • Search Google Scholar
    • Export Citation
  • XuX De Pergola G & Bjorntorp P 1990 The effects of androgens on the regulation of lipolysis in adipose precursor cells. Endocrinology1261229–1234.

    • Search Google Scholar
    • Export Citation
  • ZumoffB Strain GW Miller LK Rosner W Senie R Seres DS & Rosenfeld RS 1990 Plasma free and non-sex-hormone-binding-globulin-bound testosterones are decreased in obese men in proportion to their degree of obesity. Journal of Clinical Endocrinology and Metabolism71929–931.

    • Search Google Scholar
    • Export Citation

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    Overview of the effects of dihydrotestosterone on energy substrate pathways and adipocyte differentiation in retroperitoneal adipose tissue. Abbreviations: ACADVL, acyl-coenzyme A dehydrogenase very long chain; ACLY, ATP citrate lyase; ACTβ, actin beta cytoplasmic; ALDO1, aldolase 1 A isoform; APOE, apolipoprotein E; CCNI, cyclin I; C/EBPα, CCAAT/enhancer binding protein alpha; CES3, carboxylesterase 3; chol, cholesterol; COL1α2,3α1,4α1,5α3,6α1, procollagen type I alpha 2, type III alpha 1, type IV alpha 1, type V alpha 3, type VI alpha 1; CPT2, carnitine palmitoyltransferase 2; DDX5, DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 5; DGAT1, diacylglycerol O-acyltransferase 1; ENO1, enolase 1 alpha nonneuron; ECM, extracellular matrix; FA, fatty acids; FABP4, adipocyte fatty acid-binding protein 4; FAE, fatty acyl elongase; FAS, fatty acid synthase; G0S2, G0/G1 switch gene 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHR, growth hormone receptor; GPD1, glycerol-3-phosphate dehydrogenase 1 (soluble); GSN, gelsolin; HSL, hormone-sensitive lipase; KRT1,5,6 g,14, keratin genes 1, 5, 6 g and 14; LDH1, lactate dehydrogenase 1 A chain; LPL, lipoprotein lipase; LRP1, low-density lipoprotein receptor-related protein 1; ME, malic enzyme supernatant; MGL, monoglyceride lipase; MYH4, myosin heavy polypeptide 4 skeletal muscle; MMP2, matrix metalloproteinase 2; nascent-lp, nascent lipoprotein; PCX, pyruvate carboxylase; PGAM1, phosphoglycerate mutase 1; SCD1, stearoyl-coenzyme A desaturase 1; SLC25, solute carrier family 25 (mitochondrial carrier; citrate transporter) member 1; SPARC, secreted acidic cysteine-rich glycoprotein; TALDO1, transaldolase 1; TG, triglyceride; TKT, transketolase; TPI, triosephosphate isomerase; TUBα1,2,6, EST tubulin alpha 1, 2 and 6; VLDL, very low-density lipoprotein. *Except one EST.

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    • Search Google Scholar
    • Export Citation
  • LashAE Tolstoshev CM Wagner L Schuler GD Strausberg RL Riggins GJ & Altschul SF 2000 SAGEmap: a public gene expression resource. Genome Research101051–1060.

    • Search Google Scholar
    • Export Citation
  • LiaoX Thrasher JB Pelling J Holzbeierlein J Sang QX & Li B 2003 Androgen stimulates matrix metalloproteinase-2 expression in human prostate cancer. Endocrinology1441656–1663.

    • Search Google Scholar
    • Export Citation
  • MaK Cabrero A Saha PK Kojima H Li L Chang BH Paul A & Chan L 2002 Increased beta-oxidation but no insulin resistance or glucose intolerance in mice lacking adiponectin. Journal of Biological Chemistry27734658–34661.

    • Search Google Scholar
    • Export Citation
  • MackallJC Student AK Polakis SE & Lane MD 1976 Induction of lipogenesis during differentiation in a ‘preadipocyte’ cell line. Journal of Biological Chemistry2516462–6464.

    • Search Google Scholar
    • Export Citation
  • MarinP1995 Testosterone and regional fat distribution. Obesity Research3 (Suppl 4) 609S–612S.

  • MarinP Krotkiewski M & Bjorntorp P 1992 Androgen treatment of middle-aged obese men: effects on metabolism muscle and adipose tissues. European Journal of Medicine1329–336.

    • Search Google Scholar
    • Export Citation
  • MarinP Oden B & Bjorntorp P 1995 Assimilation and mobilization of triglycerides in subcutaneous abdominal and femoral adipose tissue in vivo in men: effects of androgens. Journal of Clinical Endocrinology and Metabolism80239–243.

    • Search Google Scholar
    • Export Citation
  • MironovAA Fickett JW & Gelfand MS 1999 Frequent alternative splicing of human genes. Genome Research91288–1293.

  • NakajimaI Muroya S Tanabe R & Chikuni K 2002 Extracellular matrix development during differentiation into adipocytes with a unique increase in types V and VI collagen. Biology of the Cell94197–203.

    • Search Google Scholar
    • Export Citation
  • NelsonCC Hendy SC Shukin RJ Cheng H Bruchovsky N Koop BF & Rennie PS 1999 Determinants of DNA sequence specificity of the androgen progesterone and glucocorticoid receptors: evidence for differential steroid receptor response elements. Molecular Endocrinology132090–2107.

    • Search Google Scholar
    • Export Citation
  • NovakJP Sladek R & Hudson TJ 2002 Characterization of variability in large-scale gene expression data: implications for study design. Genomics79104–113.

    • Search Google Scholar
    • Export Citation
  • OishiM Taniguchi Y Nishimura K Yamada T & Sasaki Y 2000 Characterisation of gene expression in bovine adipose tissue before and after fattening. Animal Genetics31166–170.

    • Search Google Scholar
    • Export Citation
  • PauwsE van Kampen AH van de Graaf SA de Vijlder JJ & Ris-Stalpers C 2001 Heterogeneity in polyadenylation cleavage sites in mammalian mRNA sequences: implications for SAGE analysis. Nucleic Acids Research291690–1694.

    • Search Google Scholar
    • Export Citation
  • SchenaM Shalon D Davis RW & Brown PO 1995 Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science270467–470.

    • Search Google Scholar
    • Export Citation
  • Schuilenga-HutPH Vlies P Jonkman MF Waanders E Buys CH & Scheffer H 2003 Mutation analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa simplex and identification of novel mutations. Human Mutations21447.

    • Search Google Scholar
    • Export Citation
  • SeidellJC2000 Obesity insulin resistance and diabetes – a worldwide epidemic. British Journal of Nutrition83 (Suppl 1) S5–8.

  • SeidellJC Bjorntorp P Sjostrom L Kvist H & Sannerstedt R 1990 Visceral fat accumulation in men is positively associated with insulin glucose and C-peptide levels but negatively with testosterone levels. Metabolism39897–901.

    • Search Google Scholar
    • Export Citation
  • SheehanMT & Jensen MD 2000 Metabolic complications of obesity. Pathophysiologic considerations. Medical Clinics of North America84363–385 vi.

    • Search Google Scholar
    • Export Citation
  • SjostromL Smith U Krotkiewski M & Bjorntorp P 1972 Cellularity in different regions of adipose tissue in young men and women. Metabolism211143–1153.

    • Search Google Scholar
    • Export Citation
  • St-AmandJ Okamura K Matsumoto K Shimizu S & Sogawa Y 2001 Characterization of control and immobilized skeletal muscle: an overview from genetic engineering. FASEB Journal15684–692.

    • Search Google Scholar
    • Export Citation
  • TsaiEC Boyko EJ Leonetti DL & Fujimoto WY 2000 Low serum testosterone level as a predictor of increased visceral fat in Japanese-American men. International Journal of Obesity-Related Metabolic Disorders24485–491.

    • Search Google Scholar
    • Export Citation
  • VagueJ1956 The degree of masculine differentiation of obesities: a factor determining predisposition to diabetes atherosclerosis gout and uric calculous disease. American Journal of Clinical Nutrition420–34.

    • Search Google Scholar
    • Export Citation
  • VelculescuVE Zhang L Vogelstein B & Kinzler KW 1995 Serial analysis of gene expression. Science270484–487.

  • VelculescuVE Zhang L Zhou W Vogelstein J Basrai MA Bassett DE Jr Hieter P Vogelstein B & Kinzler KW 1997 Characterization of the yeast transcriptome. Cell88243–251.

    • Search Google Scholar
    • Export Citation
  • VirlonB Cheval L Buhler JM Billon E Doucet A & Elalouf JM 1999 Serial microanalysis of renal transcriptomes. PNAS9615286–15291.

  • WeinerFR Smith PJ Wertheimer S & Rubin CS 1991 Regulation of gene expression by insulin and tumor necrosis factor alpha in 3T3-L1 cells. Modulation of the transcription of genes encoding acyl-CoA synthetase and stearoyl-CoA desaturase-1. Journal of Biological Chemistry26623525–23528.

    • Search Google Scholar
    • Export Citation
  • XuX De Pergola G & Bjorntorp P 1990 The effects of androgens on the regulation of lipolysis in adipose precursor cells. Endocrinology1261229–1234.

    • Search Google Scholar
    • Export Citation
  • ZumoffB Strain GW Miller LK Rosner W Senie R Seres DS & Rosenfeld RS 1990 Plasma free and non-sex-hormone-binding-globulin-bound testosterones are decreased in obese men in proportion to their degree of obesity. Journal of Clinical Endocrinology and Metabolism71929–931.

    • Search Google Scholar
    • Export Citation