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Endocrinology, doi:10.1210/en.2007-1151
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Endocrinology Vol. 149, No. 4 1551-1561
Copyright © 2008 by The Endocrine Society

Gene Expression Analyses in Cynomolgus Monkeys Provides Mechanistic Insight into High-Density Lipoprotein-Cholesterol Reduction by Androgens in Primates

Pascale Nantermet1, Shun-ichi Harada1,2, Yuan Liu, Spring Cheng, Colena Johnson, Yuanjiang Yu, Donald Kimme, Daniel Holder, Paul Hodor, Robert Phillips and William J. Ray

Departments of Molecular Endocrinology and Bone Biology (P.N., S.-i.H., Y.Y., D.K., W.J.R.), Laboratory Animal Resources (C.J.), and Biometrics (D.H.), Merck Research Laboratories, West Point, Pennsylvania 19486; and Department of Molecular Profiling (Y.L., S.C., P.H., R.P.), Rosetta Inpharmatics LLC, Seattle, Washington 98109

Address all correspondence and requests for reprints to: William J. Ray, Department of Alzheimer’s Disease Research, Merck Research Laboratories, West Point, Pennsylvania 19486. E-mail: james_ray{at}merck.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens increase muscle mass, decrease fat mass, and reduce high-density lipoprotein cholesterol (HDL), but the relationship between body composition, lipoprotein metabolism, and androgens has not been explained. Here we treated ovariectomized cynomolgus monkeys with 5{alpha}-dihydrotestosterone (DHT) or vehicle for 14 d and measured lipoprotein and triglycerides. Nuclear magnetic resonance analysis revealed that DHT dose-dependently reduced the cholesterol content of large HDL particles and decreased mean HDL particle size (P < 0.01) and also tended to lower low-density lipoprotein cholesterol without altering other lipoprotein particles. Liver and visceral fat biopsies taken before and after DHT treatment for 1 or 14 d were analyzed by genome-wide microarrays. In liver, DHT did not alter the expression of most genes involved in cholesterol synthesis or uptake but rapidly increased small heterodimer partner (SHP) RNA, along with concomitant repression of CYP7A1, a target of SHP transcriptional repression and the rate-limiting enzyme in bile acid synthesis. DHT regulation of SHP and CYP7A1 also occurs in rats, indicating a conserved mechanism. In adipose tissue, pathway analyses suggested coordinate regulation of adipogenesis, tissue remodeling, and lipid homeostasis. Genes encoding IGF-I and β-catenin were induced, as were extracellular matrix, cell adhesion, and cytoskeletal components, whereas there was consistent down-regulation of genes involved in triacylglycerol metabolism. Interestingly, cholesterol ester transfer protein RNA was induced rapidly in monkey adipose tissue, whereas its inhibitor apolipoprotein CI was repressed. These data provide insight into the androgenic regulation of lipoprotein homeostasis and suggest that changes in adipose lipoprotein metabolism could contribute to HDL cholesterol reduction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEN ARE MORE prone to cardiovascular disease than age-matched premenopausal women and concomitantly have lower circulating levels of antiatherogenic high-density lipoprotein (HDL)-cholesterol (C). Because androgen administration lowers HDL-C in both genders (1) particularly at supraphysiological plasma concentrations, endogenous androgens such as testosterone have been implicated in these gender differences in lipoprotein profile and cardiovascular disease (CVD) risk. However, the relationship between androgens and CVD risk factors is highly complex and often contradictory (reviewed in Ref. 2). Several studies report an association between low circulating androgens and low HDL-C in both men (3, 4) and women (5), and in other studies androgen depletion by castration or gonadotropin treatment increased low-density lipoprotein (LDL) without affecting HDL (6, 7). Genetic studies examining the CAG repeat length in the androgen receptor (AR), in which shorter repeat lengths are associated with increased androgen signaling, generally agree that the more active, shorter repeat length receptor is associated with lower HDL-C (8, 9) but does not influence risk for CVD (10, 11). Furthermore, low testosterone has been associated with increased risk for CVD in multiple cross-sectional studies (12). Because natural and synthetic androgens are used or are being developed as therapies for sarcopenia, osteoporosis, low libido, male contraception, and many other conditions, it will be important to understand their effects on CVD risk in the various target populations. However, to date there have been no large controlled studies examining the cardiovascular risks and benefits associated with androgen use (13). A beneficial effect of androgens was observed in rabbits and mice with experimentally induced atherosclerosis (14, 15), but the relevance of these models for steroid hormone effects on the human cardiovascular system is unclear.

A likely explanation for the complex relationship between androgens and CVD is that androgens affect many risk factors. For example, androgens can increase muscle mass, decrease visceral fat mass in some subjects, improve coronary blood flow, increase mood and motivation (perhaps leading indirectly to health benefits), are in some regards antiinflammatory, reduce lipoprotein (a) and leptin, improve insulin sensitivity, and provide other potential benefits (16). Furthermore, the reduction of HDL-C observed with androgen administration is also not necessarily an indication of elevated risk for atherosclerosis because it could indicate enhanced reverse cholesterol transport. To better understand the role of endogenous and therapeutic androgens in CVD, it will be necessary to identify the mechanisms responsible for the changes in HDL-C and how it relates to the other physiological effects. One proposed mechanism is the induction of hepatic lipase, which remodels lipoprotein particles and could account for the reduction in HDL-C (17). This hypothesis is consistent with the selective reduction of large HDL particles found in some studies (17, 18). However, it is not known whether hepatic lipase induction is a cause or consequence of HDL-C reduction, and in a single male subject with congenital HL deficiency, the androgen stanozolol caused profound reductions in HDL-C (19). In some, but not all, studies, apolipoprotein-AI (Apo-AI) levels are reduced after androgen treatment, suggesting decreased synthesis or increased catabolism of this core constituent of HDL particles (18, 20). These observations are intriguing but have not been uniformly observed and lack mechanistic explanation.

Another possibility that has not been explored is that androgens alter lipoprotein metabolism secondary to changes in lipid homeostasis in adipose tissue. The AR is highly expressed in adipocytes and regulates their function by a variety of mechanisms, including local transcriptional regulation of lipases and increased levels of β-adrenergic receptors as well as inhibition of adipogenesis (21). The AR is also expressed in liver, a primary site of lipoprotein regulation, in which it could conceivably alter the expression of genes controlling HDL metabolism. However, studies identifying AR target genes in the liver are lacking. Because AR is a ligand-activated transcription factor, androgens exert their influence primarily by altering the rate of transcription of target genes, making genome-wide microarray gene expression studies ideal for determining the primary mechanisms underlying physiological changes.

To gain a better understanding of the possible effect of androgen on cholesterol metabolic processes, we analyzed the effects of 5{alpha}-dihydrotestosterone (DHT). DHT is a potent natural androgen that, unlike testosterone, cannot be converted to estradiol by aromatase and thus acts selectively on the AR. Cynomolgus monkeys are validated models for atherosclerosis (22) and offer the advantage of expressing key lipid regulatory genes such as CETP, which rodents do not, and more closely mirroring human LDL to HDL ratios than rodents. In contrast to intact females, ovariectomized females will not experience changing endogenous estrogen levels, which could make data interpretation difficult, due to either menstrual status or secondary to LH suppression by experimentally administered androgens. Furthermore, premenopausal cynomolgus monkeys appear relatively protected against diet-induced atherosclerosis, and this effect is lost on ovariectomy (23), suggesting that they are suitable models for studying the effects of sex steroids on cardiovascular risk factors. Here we report the effects of DHT on lipoprotein levels and gene expression in liver and visceral adipose tissue in ovariectomized cynomolgus monkeys.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All procedures performed with animals were in accordance with established guidelines and were reviewed and approved by the Institutional Animal Care and Use Committee. Cynomolgus monkeys (Macaca fascicularis) from Mauritius origin were individually housed and cared for in a facility that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animals were fed a commercially prepared diet (high protein monkey diet 5045; PMI Nutritional International, St. Louis, MO). Water was offered ad libitum.

Two studies were conducted in the same cohort, with 10 month’s time intervening. For test article administration, animals were chair restrained for 30 min. All animals were fasted before specimen collection. In both studies randomized subjects (n = 5–6/group) were injected sc with vehicle (13% benzyl alcohol in cotton seed oil, 0.5 ml/kg; sc) for 7 d. On the seventh day, blood was drawn and liver and visceral adipose tissue biopsied by laparoscopy. The next day animals were treated daily with vehicle or DHT for 14 d. The day of the last injection, the second blood collection and liver and adipose biopsies were conducted. Whole blood was prepared as serum and biopsy specimens were frozen in liquid nitrogen. In one study (termed study 2 in the figures), body composition was measured by whole-body dual x-ray absorptiometry (DEXA) on the days of sample collection and on the 13th day of treatment. In the other study (termed study 1), two animals from each of the three DHT dose groups were also used to collect serum for pharmacokinetic data over a 24-h period (Fig. 1CGo).


Figure 1
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  		FIG. 1. Anabolic effects of DHT. Androgen-induced changes in body composition in ovariectomized cynomolgus monkeys (n = 5/group). Animals were pretreated with vehicle for 7 d and then received either 2.5 mg/kg DHT or vehicle for 2 additional weeks. DEXA measurements of Lean body (A) and fat mass (B) were taken just before treatment and immediately at the end of the 2-wk period. *, Significantly (P ≤ 0.05) different from vehicle control (Student’s t test) ± SE. C, Pharmacokinetic analysis of serum DHT concentrations 13 d after DHT administration at the indicated dose (n = 2; error bars represent range of values).

 
Additionally, in separate animals that were naïve to androgen treatment, we conducted an experiment with intact, aged (15–20 yr old), female cynomolgus monkeys (n = 2) with a single, high-dose DHT (25 mg/kg). The animals were from the same origin and provider and were housed as before. Briefly, the animals were treated with vehicle for 7 d. After blood collection and liver and both visceral and dorsal sc adipose tissue biopsy, animals were injected with DHT or vehicle, and 24 h later, a second posttreatment biopsy was collected.

DHT measurements
DHT was measured in serum samples by radioimmune assay following the manufacturer’s guidelines (Diagnostic Systems Laboratories, Webster, TX). The antibody used to detect DHT is >50-fold selective against androstenedione and estradiol, and 5000-fold selective against testosterone (Diagnostic Systems Laboratories, Webster, TX; product insert). Standard curves were generated by spiking DHT into plasma from an untreated ovariectomized female Cynomolgus monkey; and in this matrix the lower limit of reliable quantification was 3.2 nM. Reliably quantifiable signal above this level was present only in plasma samples from animals that had been dosed with 0.25 or 2.5 mg/kg DHT (Fig. 1Go).

Rats
Sprague Dawley female rats (Taconic, Germantown, NY) were ovariectomized at the age of 3 months and treated at the age of 7 months. Animals were randomized into eight groups (n = 10), with four groups receiving vehicle alone (propylene glycol), and four groups receiving 3 mg/kg DHT. All animals were treated daily by sc injections for 4, 7, 10, or 17 d, fasted overnight, and killed using CO2. Livers were frozen in liquid nitrogen for RNA analysis.

Cholesterol and Apo-AI measurements
The serum analysis of total cholesterol, HDL-C, LDL-C, and triglyceride were performed by Lipomed, Inc. (Raleigh, NC) by nuclear magnetic resonance. Serum Apo-A1 protein was measured by ELISA according to the manufacture (Sigma, St. Louis, MO).

Total RNA and RT-PCR
Total RNA was extracted using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). Subsequently the samples were DNase I treated with RNeasy/DNase I as directed by the manufacturer (QIAGEN, Santa Clarita, CA). Quantitative RT-PCR was carried out using Brilliant one step RT-PCR kit and the MX4000/MX3000 sequence detection system according to the manufacturer’s protocols (Stratagene, La Jolla, CA). Then 75-ng aliquots of RNA were reverse transcribed and amplified using the following primers and Taqman probes (Tables 1Go and 2Go). ABCA1 primers and probe were purchased from Applied Biosystems (Foster City, CA). Data were normalized to HS10 for the nonhuman primate and to 18S RNA (primers and probes from Applied Biosystems) for the rodent. Average cycle threshold (Ct) values from duplicate PCR were normalized to average Ct values for HS10 or 18S from the same cDNA preparations. The ratio of expression of each gene in experimental vs. control samples was calculated as 2–(mean{Delta}{Delta}Ct). Significant differences were determined using ANOVA.


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  		TABLE 1. Primers and probes used for cynomolgus monkey RT-PCR

 

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  		TABLE 2. Primers and probes used for rat RT-PCR

 
Microarray
Total RNA isolated from monkey tissues was converted into fluorescence-labeled cRNA that was hybridized to DNA oligonucleotide microarrays as described previously (24). Briefly, 5 µg of total RNA from an individual sample were used to synthesize double-stranded DNA by RT. cRNA was produced by in vitro transcription and posttranscriptionally labeled with either Cy3 or Cy5. Reference and experimental cRNA samples were competitively hybridized to Functional ID version 2.0 human 25K arrays (Agilent Technologies, Palo Alto, CA). To minimized bias created by dye selection, for each comparison two hybridizations were performed with each cRNA sample pair using a fluorescent dye reversal strategy. After hybridization, arrays were scanned and fluorescence intensities for each probe were recorded. Ratios of transcript abundance in experimental vs. control samples were calculated with normalized intensity data. Gene expression data analysis was conducted either with the Rosetta Resolver gene expression analysis software (version 5.1; Rosetta Biosoftware, Seattle, WA) or Matlab (version 7,; The Mathworks, Natick, MA). For each gene sequence on the arrays, statistical significance of differential gene expression was calculated according to the following equation:

Formula
where Erf is the error function for a Gaussian distribution of zero mean and xdev is the adjusted difference in fluorescence intensities between Cy3 and Cy5 intensities as calculated by the equation:

Formula
where r is the Cy5 intensity, g is Cy3 intensity, and {sigma} is the error associated with the respective channel. The analyzed microarray data are included as supplemental data, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lean and fat body mass composition after DHT treatment
To evaluate the effect of androgens on body composition and lipid metabolism in surgically menopausal female cynomolgus monkeys, we first sought to identify an effective dose range. Pilot studies suggested that 2.5 mg/kg DHT, administered sc in 13% benzyl alcohol and sesame oil, stimulated bone formation and reduced HDL-C (the latter effect occurring within 4 d) (data not shown). Thus, after 7 d of vehicle treatment to establish a baseline condition, surgically menopausal female cynomolgus monkeys (n = 6 per group) were randomized to receive 2.5 mg/kg DHT or vehicle daily to for 2 wk. The lean body mass and fat mass were measured by DEXA after the 7-d vehicle regimen and on the last day of DHT dosing, and serum samples were collected for lipoprotein analysis on these same days (see below). The animals gained an average of 745 g of lean body mass (P < 0.05, ANOVA) on DHT administration (Fig. 1AGo), which corresponded to a significant reduction in percent fat mass (Fig. 1BGo). These data indicate that DHT improves lean body mass and body composition in ovariectomized cynomolgus monkeys and established an effective dose for further experimentation.

To understand the dose-exposure relationship, a multiple-dose pharmacokinetic study using 2.5, 0.25, and 0.025 mg/kg DHT was conducted. After 13 d of treatment, blood was collected at varying times up to 24 h after the dose, and DHT levels were measured by radioimmune assay (Fig. 1CGo). The dose of 2.5 mg/kg DHT produced a mean maximal serum concentration of 82 nM (n = 2) and sustained serum levels over 20 nM for the entire 24-h period. The 0.25 mg/kg DHT dose generated lower serum concentrations (maximal concentration of 18 nM, n = 2) that were maintained in the 10-nM range over several hours. The 0.025 mg/kg DHT dose did not generate DHT levels above the lower limit of reliable quantification (3.2 nM).

Serum level of total cholesterol, HDL, LDL, and very low-density lipoprotein (VLDL) cholesterol
To analyze the effect of androgens on lipoprotein metabolism, we measured the serum levels of lipids before and after administration of DHT (n = 6/group). The treatment consisted of sc injection of vehicle for 7 d to establish a baseline condition, followed by randomization to receive vehicle or DHT at various doses (0.025, 0.25, and 2.5 mg/kg) for a period of 2 wk. Serum was collected before and after the 14-d treatment, analyzed by nuclear magnetic resonance, and the change in parameters during the treatment period compared with the vehicle-treated group and analyzed by ANOVA. The graphs in Figs. 2–4GoGoGo represent each treatment group’s change from baseline, subtracted from the vehicle-treated animals’ change from baseline (± 95% confidence interval). Additionally, data from the study described in the previous section, which was carried out independently with the same animals randomized differently, were used as replication data and were referred to as study 2. As shown in Fig. 2AGo, 2.5 mg/kg DHT reduced total serum cholesterol (P < 0.01) in the dose-ranging study but not significantly in study 2. However, in both studies, there was a 25- to 30-mg/dL decrease in HDL-C (P = 0.01 and < 0.01, respectively, Fig. 2BGo), which appeared dose dependent. In both studies there tended to be a decrease in LDL-C but was significant only at the 2.5-mg/kg dose in one study (P = 0.02 and 0.06, Fig. 2CGo). Finally, whereas there was a trend toward VLDL-C reduction in some treatment groups, the overall effect was variable in study 2 and did not replicate (Fig. 2DGo). In both studies serum triglycerides were not significantly affected by treatment (data not shown). Thus, DHT produced a reproducible reduction in HDL-C and tended to reduce LDL-C.


Figure 2
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  		FIG. 2. Lipid profile after DHT treatment. Serum was collected before DHT treatment was initiated and at the end of the treatment. Animals (n = 5–6/group) were treated with 0.025, 0.25, or 2.5 mg/kg DHT for 2 wk, or in a separate study, 2.5 mg/kg DHT for 2 wk (study 2). Total cholesterol, HDL-C, LDL-C, and VLDL-C were measured. Data are presented as change from baseline measurement per individual. Closed circle represents the median for each group, ± 95% confidence intervals; P value was determined by ANCOVA.

 

Figure 3
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  		FIG. 3. Analysis of lipoprotein subfractions. Each fraction of cholesterol particle collected from serum was analyzed by nuclear magnetic resonance. Animals (n = 5–6/group) were treated with 0.025, 0.25, or 2.5 mg/kg DHT for 2 wk, or in a separate study, 2.5 mg/kg DHT for 2 wk (study 2). Data are shown as change from the baseline measurement. A–C, Median particle sizes in nanomoles of HDL, LDL, and VLDL, respectively. D and E, Two groups of HDL subfractions, with the larger fractions (3–5) grouped separately. F, L3 of the LDL subclass, in which L3 corresponds to the large LDL. Closed circle represents the median for each group, ± 95% confidence intervals; P value was determined by ANCOVA.

 

Figure 4
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  		FIG. 4. Serum Apo-A1. Serum Apo-A1 protein was measured by ELISA in ovariectomized cynomolgus monkeys in the dose-response study (study 1). The results represent the changes between before and after androgen treatment. Closed circle represents the median for each group, ± 95% confidence intervals; P value was determined by ANCOVA.

 
Analysis of lipoprotein particle size
We then analyzed the amount of cholesterol associated with individual lipoprotein subfractions by nuclear magnetic resonance. To clarify presentation of the data, LDL was grouped into two categories, small (LDL subfractions 1 and 2) and large (subfraction 3), and HDL was grouped likewise into small (subfractions 1 and 2) and large (subfractions 3–5). The data were assessed by analysis of covariance (ANCOVA) and is graphed as group means (n = 5–6/group) of changes from baseline subtracted from vehicle effects (± 95% confidence interval). DHT produced a dose-dependent reduction in mean HDL particle size ranging from 0.5–1.0 nm (P < 0.01 in both studies for 2.5 mg/kg DHT, Fig. 3AGo) and tended to decrease LDL particle size, reaching significance only in study 2 (Fig. 3BGo). There was no effect on VLDL size (Fig. 3CGo). Consistent with this observation, the larger particles of HDL were specifically reduced approximately 30 mg/dl, accounting for the reduction in total HDL-C (Fig. 3Go, D and E). Larger LDL particles again tended to be reduced but did not replicate consistently (Fig. 3FGo), and no effect on other particles was observed (data not shown). We examined the serum levels of Apo-A1 by ELISA in the same samples. In this particular study, no significant change in serum Apo-A1 was observed (Fig. 4Go), although in some pilot studies, Apo-A1 was repressed (data not shown). Taken together, DHT treatment for 2 wk increased lean body mass and decreased percent fat mass, which was accompanied by a decrease in mean HDL particle size and the cholesterol content of large HDL particles.

Microarray analysis
To understand the effect of DHT at the genomic level and identify potential mechanisms underlying the effects on HDL-C large particles, microarray analysis was performed in the liver and visceral adipose tissue specimens of study 1. Briefly, biopsies were taken before and after treatment, and tissues were frozen in liquid nitrogen for RNA extraction. RNA was examined for integrity, and two vehicle treatment group liver specimens and one visceral adipose tissue specimen from each treatment group were excluded on this basis. The baseline and posttreatment RNA samples were then labeled with either Cy3 or Cy5 fluorescent dyes, and samples from the same individual monkey were mixed and competitively hybridized to 25,000 feature human microarrays. This strategy allows the detection of genes that changed as a response to treatment within an individual animal. Fluorescent signals were background adjusted and analyzed. The ratio of posttreatment to baseline expression values was used as the numerical value assigned to each transcript, and the data were analyzed for significant treatment effects by ANOVA. Transcripts were selected if the following criteria were met: the median hybridization intensity was above nonspecific background, differed from vehicle significantly (P < 0.05), and was 1.2-fold different from vehicle. Based on those criteria, 667 and 1477 transcripts were identified, respectively, in liver and visceral fat, respectively (Fig. 5Go, A and B).


Figure 5
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  		FIG. 5. Microarray overview in liver and visceral adipose tissues 2 wk after DHT treatment. 1-D ordered agglomerative clustering of the regulated gene set was performed using Resolver software. Each row is a different animal, and each column is a false-color map of a single gene’s expression relative to vehicle in log10 scale. Below each heat map the number of transcripts is recorded. A, Liver biopsies after 2 wk treatment with 2.5 mg/kg DHT. B, Visceral adipose cluster after 2 wk treatment with 2.5 mg/kg DHT.

 
In a second study designed to determine primary response genes from secondary effects, intact female cynomolgus monkeys were administered a single high-dose of DHT (25 mg/kg) or vehicle (n = 2). This high dose was chosen to ensure robust responses of genes that rapidly respond to activation of the AR. Furthermore, pilot studies indicated that HDL-C reduction by androgens requires 4 d of dosing (data not shown), so the genes regulated by DHT within this time frame are less likely to be responding secondary to the changes in cholesterol metabolism. RNA prepared from biopsies from liver, sc fat, and visceral fat were subjected to microarray analyses as above. One vehicle treatment group sample from the sc adipose site was disqualified. Due to the low number of samples, a simple analysis scheme was used to enrich for potentially regulated genes. Transcripts were selected for further consideration if both DHT treatment samples but neither vehicle sample produced significant change in fluorescent hybridization intensities relative to baseline (P < 0.05) as judged by the Rosetta error model, and the mean fold change was greater than 1.5. A total of 240 transcripts were selected on this basis from liver and 1004 transcripts were selected from visceral fat. Regulated transcripts from each microarray experiment were analyzed by one-dimensional agglomerative clustering, such that the order of the samples are fixed and the genes are grouped by correlation coefficient and are displayed in Figs. 5Go and 6Go.


Figure 6
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  		FIG. 6. Microarray overview in liver and adipose tissues after 24 h high dose of DHT. 1-D ordered agglomerative clustering of the regulated gene set was performed using Resolver software. Each row is a single animal; each column is a false-color map of a single gene’s expression relative to vehicle in log10 scale. Below each heat map the number of transcripts is recorded. A, Liver biopsies before and after 25 mg/kg DHT. B, Adipose tissue (sc and visceral fat) biopsies before and after 24 h 25 mg/kg DHT.

 
Analysis of liver microarray data
Changes in gene expression in liver were first scrutinized for genes with known functions in the regulation of cholesterol or lipoprotein homeostasis. At both time points, there was no regulation of the genes encoding Apo-AI, scavenger receptor type B, class I, 3-hydroxy-3-methylglutaryl coenzyme A reductase, the LDL receptor, or most of the many genes known to participate in these pathways (data not shown). Regulated transcripts were submitted to GeneGo, a pathway analysis software tool that identifies overrepresentation of functional terms in a list of genes relative to the whole genome. After correction for multiple testing, the P value for enrichment of the term cholesterol metabolism in the 24 h data set was 1.0, and in the 2-wk data set was 0.92. Three genes with known roles in cholesterol synthesis were induced at 2 wk: squalene epoxidase, cyp51a1, and 24-dehydrocholesterol reductase. However, manual inspection of the data did not reveal any other genes in the cholesterol synthesis pathway with consistent trends toward induction. Although not reaching significance by unbiased pathway analysis, we noted the modest induction of three genes with known roles in lipoprotein metabolism: hepatic lipase, a secreted lipase induced by androgen treatment (25, 26); Apo-AII, an HDL-associated lipoprotein; and Apo-CIII, a VLDL component (Fig. 7AGo). Pathway analysis did detect significant enrichment in three pathways, including a uniform induction of nine genes involved in mitochondrial oxidative phosphorylation, particularly in complex I (Fig. 7BGo). Likewise, pathway analysis of the 1-d treatment data revealed four significant term enrichments, including the modulation of growth factor signaling pathways such as IGF-I, which is known to be modulated by androgens (27) (Fig. 7CGo).


Figure 7
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  		FIG. 7. Analysis of the liver microarray data. A, Microarray data for the RNAs encoding hepatic lipase, apolipoprotein-AII (APOA2), and apolipoprotein-CIII (APOC3), mean values ± SD. *, P < 0.05 ANOVA. Bottom, GeneGo pathway analysis of the entire regulated data set at 14 d (B) and 24 h (C) after treatment, showing significant findings (P < 0.05 after multiple test correction, giving the expectation score). Overlap Gene Count is the number of genes in the data set annotated with the indicated pathway term, of a possible set in the whole genome (Set Gene Count). The Input Identifiers are the official Entrez Gene abbreviations for the identified genes.

 
Small heterodimer partner (SHP) and CYP7A1 in the liver
Further mining of the data led to the observation that in both studies DHT induced the RNA for SHP (NR0B2), which acts as a transcriptional corepressor of cholesterol 7{alpha}-hydroxylase (CYP7A1) expression, the rate-limiting enzyme in bile acid biosynthesis (28), and concomitantly CYP7A1 RNA was repressed. To confirm this observation, quantitative RT-PCR was conducted on the RNA samples used in the microarray and showed that SHP was induced approximately 3-fold at both time points (P < 0.05), whereas CYP7A1 was repressed approximately 3-fold at the 2-wk time point (Fig. 8AGo). SHP and CYP7A1 regulation is species specific (29); thus, we measured the expression of these genes in the livers of ovariectomized female rats treated for 4, 7, 10, or 17 d with daily sc injection of 3 mg/kg DHT or vehicle. This treatment regimen produces a decrease in serum HDL-C levels in this animal model (data not shown) and, as in the cynomolgus monkeys, caused a significant increase in SHP of approximately 3-fold at all time points, whereas Cyp7a1 was similarly repressed (Fig. 8BGo). Thus, in two species, DHT caused a rapid and sustained induction of SHP and repression of CYP7A1 RNA.


Figure 8
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  		FIG. 8. Conservation of SHP and CYP7A1 across species. A, Quantitative RT-PCR analysis of SHP and CYP7A1 RNA expression relative to vehicle (Veh) in the liver biopsies. All values were normalized to HS10 housekeeping gene RNA to control for RNA input. Shown are means ± SD. *, P value <0.05 (ANOVA). B, Rats (n = 10/group) ovariectomized at 3 months of age were treated for the indicated numbers of days with vehicle or 3 mg/kg DHT. Total RNA was extracted from liver and subjected to quantitative RT-PCR. Shown are the relative expression to vehicle for two key genes involved in cholesterol metabolism and bile acid conversion. The data were normalized to 18S RNA, and the vehicle was arbitrary set to 1. Shown are means ± SD. *, P value < 0.05 (ANOVA).

 
Analysis of gene expression data in visceral adipose tissue
The microarray gene expression data from adipose tissue were then analyzed as above. In the single high-dose experiment, we included sc fat due to the high expression of the AR in this sexually dimorphic tissue (30). Pairwise comparison showed that most of the genes regulated in visceral fat were also regulated in sc fat (Fig. 6Go), and comparison of these data revealed highly significant correlations between the two tissues (r = 0.78, P < 0.001). Thus, DHT produced similar gene expression responses in these two adipose sites.

In contrast to the liver microarray data, both manual functional clustering and software-based pathway analyses of the visceral adipose tissue data revealed many coordinated changes in gene expression, including in lipid metabolism, adipogenesis, and lipoprotein homeostasis. To confirm these regulations, we evaluated eight genes with apparent regulation by DHT using quantitative RT-PCR on the same RNA samples used for microarray. As shown in Fig. 9Go, all eight were differentially expressed as predicted from the microarray data; the potential relevance of these genes are discussed below.


Figure 9
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  		FIG. 9. Quantitative RT-PCR confirmation of selected genes in visceral fat. RNA was extracted from visceral biopsies taken prior to treatment, 24 h, and 2 wk after DHT. Shown are the relative expression at 24 h (light bars) with n = 2, and 2 wk (black bars) with n = 5–6 of RBP4, β-catenin, IGF-I, DGAT2, malic enzyme 3 (ME3), CETP, lipase, hormone-sensitive (LIPE), and ATP binding cassette transporter 1 (ABCA1). The data were normalized to HS10; the vehicle value was arbitrary set to 1 (no significant changes were seen in vehicle samples, not shown). Shown are means ± SD. *, P value < 0.05 (ANOVA).

 
Manual inspection of the data revealed that the RNAs for cholesterol ester transfer protein (CETP) was induced and apolipoprotein-CI was repressed (Fig. 10AGo); CETP induction was further confirmed by quantitative RT-PCR and was significant at both 24 h and 14 d (Fig. 9Go). Both of these regulations appeared to occur in sc adipose tissue at 24 h, with the caveat that only one vehicle-treated sample was available for comparison (Fig. 10AGo). MetaCore (GeneGo, St. Joseph, MI) analysis of the 14-d and 24-h microarray data (Fig. 10Go, B and C, respectively) indicated that many pathways were potentially modulated by DHT. Among the most significantly affected genes were those associated with the extracellular matrix, cell adhesion, and the cytoskeleton. In fact 12 of 13 significant pathways in the 24-h analysis are related to this finding (Fig. 10CGo). After 14 d of treatment, there were multiple pathways related to lipid homeostasis and energy metabolism in adipose tissue. There was consistent down-regulation of genes involved in phospholipid, fatty acid, and triacylglycerol metabolism, such as malic enzyme 3 and diacylglycerol O-acyltransferase 2 (DGAT2), which were confirmed by quantitative RT-PCR (Fig. 9Go). Hormone-sensitive lipase (LIPE), which is inhibited by androgens in human adipose tissue (31, 32), was confirmed to be repressed here (Fig. 9Go). Among the other key lipid-regulating genes that were repressed include mitochondrial glycerol 3-phosphate acyltransferase, which catalyzes the first step in glycerolipid biosynthesis and was repressed 7.1 ± 2.5-fold (data not shown). DHT also altered the expression of genes related to adipogenesis. These include the inductions of RNAs for IGF-I, a paracrine regulator of adipocyte differentiation (33), and the antiadipogenic transcription factor β-catenin (34) (Fig. 9Go). Furthermore, both peroxisomal proliferator-activated receptor-{gamma} and CCAAT/enhancer-binding protein-{alpha}, genes essential for adipogenesis, were reduced after 2 wk by 2- to 3-fold (data not shown). Finally, we selected retinal binding protein 4 (RBP4) for RT-PCR confirmation because it is an adipokine elevated in insulin-resistant states (35, 36). RBP4 was repressed in the microarray data and in the RT-PCR confirmation data at both time points. In summary, these microarray data revealed multiple potentially important gene regulations that together suggest that adipose tissue is under significant homeostatic control by androgens.


Figure 10
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  		FIG. 10. Analysis of gene expression changes in adipose tissue. CETP and apolipoprotein-CI (APOC1) RNA values from the visceral adipose (top) and sc adipose (bottom) microarray experiments at 24 h and 2 wk. Shown are means ± SD. *, P value <0.05 (ANOVA). Right, GeneGo pathway analysis of the entire regulated visceral adipose data set at 14 d (B) and 24 h (C) after treatment, showing significant findings (P < 0.05 after multiple test correction, giving the expectation score). Overlap Gene Count is the number of genes in the data set annotated with the indicated pathway term, of a possible set in the whole genome (Set Gene Count). The Input Identifiers are the official Entrez Gene abbreviations for the identified genes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Androgens modulate cholesterol metabolism in complex and poorly understood ways. Given the importance of cholesterol homeostasis for human health, it will be important to develop concepts regarding the role of androgens in cardiovascular risk. Most studies indicate that administration of natural or synthetic androgens produces a plethora of effects, some of which appear negative, such as HDL-C reduction, and others positive, such as increased lean mass and reduced visceral fat, lower total cholesterol, and improved insulin sensitivity (37). Here we administered a nonaromatizable natural androgen, DHT, to ovariectomized female cynomolgus monkeys and examined the response of lipoprotein particles, triglycerides, and gene expression in liver and adipose tissue. It is important to note that there are important differences between the studies described here and the regulation of lipoproteins by endogenous androgens. First, whereas DHT offers the experimental advantage of selectively activating the AR, the predominant androgens in many species are testosterone and the adrenal androgens, which likely would produce different effects in vivo because they are precursors for estradiol and other hormones. Furthermore, the doses used achieve plasma concentrations of DHT far in excess of the female physiological range, and thus might produce nonphysiological effects.

As shown in Figs. 2Go and 3Go, DHT led to a robust and relatively selective reduction in HDL-C. This effect was attributable nearly exclusively to a diminution of the cholesterol content and size of large HDL particles. There were variable trends for repression of both LDL-C and Apo-AI, reaching significance in some studies but not others (Figs. 2–4GoGoGo, data not shown for Apo-AI). In general these data are consistent with human data, in which exogenous androgens typically reduce HDL-C but often have mixed effects on other parameters (18, 25, 26, 38, 39). The differences in lipoprotein responsiveness between this study and certain human experiments probably relates to the varying choices of androgens (most human studies use testosterone or a synthetic androgen rather than DHT); routes of administration (oral, im, or sc); and the gender, age, and hormonal status of the subjects. It will be important to study these variables in a controlled model system, particularly the route of administration, because orally delivered androgens have been reported to have more dramatic effects on HDL than those given parentarelly (1, 2).

A selective reduction of large HDL particles could implicate several mechanisms. One hypothesis could be reduced Apo-AI synthesis or HDL production. A second possibility is that there is decreased donation of cholesterol to HDL particles from peripheral tissues, and a third is that there is increased clearance of large HDL-C. Whereas in vitro studies favor the latter scenario (40), these nonexclusive possibilities have not been fully examined in vivo. A fourth more complex possibility is that androgens lead to HDL remodeling, cholesterol redistribution, and/or changes in lipoprotein catabolism by novel mechanisms that have not been described and thus cannot rely on literature precedent for identification. Although further study will be required to resolve these possibilities, we examined the gene expression data within this context as a step toward proposing testable hypotheses.

First, in liver we did not observe transcriptional changes in the Apo-A1 or scavenger receptor type B, class I RNA, nor have we found evidence for androgen suppression of HDL biosynthesis in primary human hepatocytes or rodent livers (Nantermet, P., Y. Yu, and W. J. Ray, unpublished observations). Combined with the generally consistent finding here and in humans that androgens spare small, mostly nascent HDL particles, it is unlikely that the HDL-C reduction is solely attributable to reduced biosynthesis. We also did not find coordinated regulation of the gene targets of the cholesterol sensor sterol regulatory element-binding protein-2 such as the LDL receptor or 3-hydroxy-3-methylglutaryl coenzyme A reductase, suggesting that at the time points studied here, there was no dramatic change in hepatic cholesterol content. The expected induction of hepatic lipase was observed (Fig. 9Go) but interestingly was not evident at 24 h. These data combined with the observation that the oral androgen stanozolol still greatly reduced HDL-C in an individual with congenital hepatic lipase deficiency (19) suggest to us that hepatic lipase induction is a consequence, not a primary cause, of androgen-mediated changes in cholesterol.

Interestingly in liver tissues we observed a rapid and sustained induction of the RNA for SHP, a ligand-independent transcriptional repressor, and consistent reduction in its molecular target CYP7A1, the rate-limiting enzyme in the classical bile acid synthesis pathway (Fig. 8AGo). This effect was also seen in ovariectomized female rat livers (Fig. 8BGo), which is noteworthy because these genes are undergo species-specific regulation by liver X receptor-{alpha}, an oxysterol-activated nuclear receptor involved in cholesterol efflux (41). At present it is difficult to propose functional significance to this RNA regulation. CYP7A1 deficiency in humans causes marked hypercholesterolemia due to poor cholesterol elimination from the liver (42); however, androgens lower cholesterol. Interestingly, the testosterone metabolite androsterone can activate the farnesoid X receptor, which in turn increases expression of SHP (43). Thus, it is possible that the regulation of SHP and CYP7A1 is secondary to hepatic metabolism of androgens and does not reflect a homeostatic response to altered cholesterol uptake. Future studies measuring hepatic and fecal cholesterol and bile acid levels will be required to determine the consequences, if any, of these observations.

In contrast to the liver microarray studies, in which few cholesterol- or lipoprotein-related gene expression changes were evident, there were several genes and pathways of interest regulated by DHT in adipose tissue. Androgens have potent effects on adipocytes, altering the rate of lipolysis and lipid metabolism (21, 32, 44, 45) and inhibiting differentiation (46, 47), and AR null male mice develop late-onset obesity (48). The data collected here suggest androgens lead to tissue remodeling because at both 24 h and 14 d after DHT treatment, there was regulation of genes significantly enriched in terms associated with cell adhesion, extracellular matrix, and the cytoskeleton (Fig. 10Go). This interpretation of the data requires confirmation with ultrastructural studies. Of particular interest is the induction of IGF-I and β-catenin, the latter of which promotes myogenesis and inhibits adipogenesis (34, 49) and is recruited to the nucleus by AR in cellular models of adipogenesis (46). Furthermore, pathway analysis identified significant changes in genes related to lipid metabolism after 14 d, including coordinate repression of genes involved in triacylglycerol and lipid biosynthesis (Fig. 10Go). Together these data are consistent with the concept that androgens reduce lipid storage as well as prevent adipogenesis, likely contributing to the decreased percent fat mass observed here and in some human studies.

Of particular interest was the induction of CETP RNA as well as the repression of the RNA encoding its inhibitor Apo-CI, the latter of which was observed 24 h after DHT treatment in adipose tissue (Fig. 10Go). Although sometimes considered a hepatic enzyme, in humans, monkeys, and other nonrodent species. CETP is also synthesized by adipocytes (50, 51), and in monkeys, adipose CETP RNA levels correlate with plasma CETP levels (51). Interestingly, CETP was transgenically overexpressed in adipocytes in mice, which lack endogenous CETP, and both Apo-AI and HDL-C were reduced concomitant with a reduction in fat mass (52). Other mouse models overexpressing CETP show increased catabolism of HDL-C and enhanced uptake of cholesterol by liver, adrenals, adipose tissue, and spleen (53). Regarding the repression of Apo-CI RNA, a potentially relevant experiment was performed when CETP-expressing mice were crossed with ApoC1–/– animals (54). The resulting offspring had multiple differences in lipoproteins, including a reduction in HDL-C and HDL particle size. However, regulation of CETP alone cannot account for androgen regulation of HDL in all species because CETP is not expressed in rats, in which androgens also reduce HDL-C.

In summary, the gene expression data here extend the understanding that androgens influence the expression of genes related to adipose homeostasis, including some genes such as CETP and APOC1 that regulate HDL metabolism. Androgen effects in adipose tissue could in turn modulate lipoprotein metabolism because this mechanism has precedence. For example, HDL-C reductions are observed in obesity (reviewed in Ref. 55), in which adipose tissue uptake of HDL-C is thought to contribute (56), and in insulin-resistant states, in which elevated lipolysis in adipose tissue plays a role (reviewed in Refs. 57 and 58). The data presented here lead us to hypothesize that lipoprotein remodeling by adipose tissue contributes to changes in HDL-C by androgens.


   Acknowledgments
 
The authors thank Dave Gilberto, Stacy Bowes, Ashleigh Bone, Tamara Montgomery, Alan Savitz, Susie Jendrowski, Pete Szczerba, Marc Washington, Sharon Adamski, Michael Gentile, Patricia Masarachia, Brenda Pennypacker, Evan Opas, and Judy Pun for technical assistance.


   Footnotes
 
Disclosure Statement: All authors were employed by Merck & Co., Inc. at the time of these studies.

First Published Online January 10, 2008

1 P.N. and S.-i.H. contributed equally to this work. Back

2 S.-i.H. is deceased. Back

Abbreviations: ANCOVA, Analysis of covariance; Apo-AI, apolipoprotein-AI; AR, androgen receptor; C, cholesterol; CETP, cholesterol ester transfer protein; Ct, cycle threshold; CVD, cardiovascular disease; DEXA, dual x-ray absorptiometry; DGAT2, diacylglycerol O-acyltransferase 2; DHT, 5{alpha}-dihydrotestosterone; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LIPE, hormone-sensitive lipase; RBP4, retinal binding protein 4; SHP, small heterodimer partner; VLDL, very low-density lipoprotein.

Received August 20, 2007.

Accepted for publication January 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Somboonporn W 2006 Testosterone therapy for postmenopausal women: efficacy and safety. Semin Reprod Med 24:115–124[CrossRef][Medline]
  2. Shabsigh R, Katz M, Yan G, Makhsida N 2005 Cardiovascular issues in hypogonadism and testosterone therapy. Am J Cardiol 96:67M–72M[Medline]
  3. Wu FC, von Eckardstein A 2003 Androgens and coronary artery disease. Endocr Rev 24:183–217[Abstract/Free Full Text]
  4. Chen RY, Wittert GA, Andrews GR 2006 Relative androgen deficiency in relation to obesity and metabolic status in older men. Diabetes Obes Metab 8:429–435[CrossRef][Medline]
  5. Lambrinoudaki I, Christodoulakos G, Rizos D, Economou E, Argeitis J, Vlachou S, Creatsa M, Kouskouni E, Botsis D 2006 Endogenous sex hormones and risk factors for atherosclerosis in healthy Greek postmenopausal women. Eur J Endocrinol 154:907–916[Abstract/Free Full Text]
  6. Braga-Basaria M, Muller DC, Carducci MA, Dobs AS, Basaria S 2006 Lipoprotein profile in men with prostate cancer undergoing androgen deprivation therapy. Int J Impot Res 18:494–498[CrossRef][Medline]
  7. Xu T, Wang X, Hou S, Zhu J, Zhang X, Huang X 2002 Effect of surgical castration on risk factors for arteriosclerosis of patients with prostate cancer. Chin Med J (Engl) 115:1336–1340[Medline]
  8. Zitzmann M, Brune M, Kornmann B, Gromoll J, von Eckardstein S, von Eckardstein A, Nieschlag E 2001 The CAG repeat polymorphism in the AR gene affects high density lipoprotein cholesterol and arterial vasoreactivity. J Clin Endocrinol Metab 86:4867–4873[Abstract/Free Full Text]
  9. Zitzmann M, Gromoll J, von Eckardstein A, Nieschlag E 2003 The CAG repeat polymorphism in the androgen receptor gene modulates body fat mass and serum concentrations of leptin and insulin in men. Diabetologia 46:31–39[Medline]
  10. Hersberger M, Muntwyler J, Funke H, Marti-Jaun J, Schulte H, Assmann G, Luscher TF, von Eckardstein A 2005 The CAG repeat polymorphism in the androgen receptor gene is associated with HDL-cholesterol but not with coronary atherosclerosis or myocardial infarction. Clin Chem 51:1110–1115[Abstract/Free Full Text]
  11. Page ST, Kupelian V, Bremner WJ, McKinlay JB 2006 The androgen receptor gene CAG repeat polymorphism does not predict increased risk of heart disease: longitudinal results from the Massachusetts Male Ageing Study. Clin Endocrinol (Oxf) 65:333–339[CrossRef][Medline]
  12. Choi BG, McLaughlin MA 2007 Why men’s hearts break: cardiovascular effects of sex steroids. Endocrinol Metab Clin North Am 36:365–377[CrossRef][Medline]
  13. Rosano GM, Cornoldi A, Fini M 2005 Effects of androgens on the cardiovascular system. J Endocrinol Invest 28:32–38[Medline]
  14. Alexandersen P, Haarbo J, Byrjalsen I, Lawaetz H, Christiansen C 1999 Natural androgens inhibit male atherosclerosis: a study in castrated, cholesterol-fed rabbits. Circ Res 84:813–819[Abstract/Free Full Text]
  15. Nathan L, Shi W, Dinh H, Mukherjee TK, Wang X, Lusis AJ, Chaudhuri G 2001 Testosterone inhibits early atherogenesis by conversion to estradiol: critical role of aromatase. Proc Natl Acad Sci USA 98:3589–3593[Abstract/Free Full Text]
  16. Malkin CJ, Pugh PJ, Jones RD, Jones TH, Channer KS 2003 Testosterone as a protective factor against atherosclerosis—immunomodulation and influence upon plaque development and stability. J Endocrinol 178:373–380[Abstract]
  17. Grundy SM, Vega GL, Otvos JD, Rainwater DL, Cohen JC 1999 Hepatic lipase activity influences high density lipoprotein subclass distribution in normotriglyceridemic men. Genetic and pharmacological evidence. J Lipid Res 40:229–234[Abstract/Free Full Text]
  18. Berg G, Schreier L, Geloso G, Otero P, Nagelberg A, Levalle O 2002 Impact on lipoprotein profile after long-term testosterone replacement in hypogonadal men. Horm Metab Res 34:87–92[Medline]
  19. Bausserman LL, Saritelli AL, Herbert PN 1997 Effects of short-term stanozolol administration on serum lipoproteins in hepatic lipase deficiency. Metabolism 46:992–996[CrossRef][Medline]
  20. Dickerman RD, McConathy WJ, Zachariah NY 1997 Testosterone, sex hormone-binding globulin, lipoproteins, and vascular disease risk. J Cardiovasc Risk 4:363–366[CrossRef][Medline]
  21. De Pergola G 2000 The adipose tissue metabolism: role of testosterone and dehydroepiandrosterone. Int J Obes Relat Metab Disord 24(Suppl 2):S59–S63
  22. Weingand KW 1989 Atherosclerosis research in cynomolgus monkeys (Macaca fascicularis). Exp Mol Pathol 50:1–15[CrossRef][Medline]
  23. Clarkson TB, Adams MR, Kaplan JR, Koritnik DR 1984 Psychosocial and reproductive influences on plasma lipids, lipoproteins, and atherosclerosis in nonhuman primates. J Lipid Res 25:1629–1634[Abstract]
  24. van ’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH 2002 Gene expression profiling predicts clinical outcome of breast cancer. Nature 415:530–536[CrossRef][Medline]
  25. Tan KC, Shiu SW, Kung AW 1999 Alterations in hepatic lipase and lipoprotein subfractions with transdermal testosterone replacement therapy. Clin Endocrinol (Oxf) 51:765–769[CrossRef][Medline]
  26. Herbst KL, Amory JK, Brunzell JD, Chansky HA, Bremner WJ 2003 Testosterone administration to men increases hepatic lipase activity and decreases HDL and LDL size in 3 wk. Am J Physiol Endocrinol Metab 284:E1112–E1118
  27. Borski RJ, Tsai W, DeMott-Friberg R, Barkan AL 1996 Regulation of somatic growth and the somatotropic axis by gonadal steroids: primary effect on insulin-like growth factor I gene expression and secretion. Endocrinology 137:3253–3259[Abstract]
  28. Bavner A, Sanyal S, Gustafsson JA, Treuter E 2005 Transcriptional corepression by SHP: molecular mechanisms and physiological consequences. Trends Endocrinol Metab 16:478–488[CrossRef][Medline]
  29. Beigneux A, Hofmann AF, Young SG 2002 Human CYP7A1 deficiency: progress and enigmas. J Clin Invest 110:29–31[CrossRef][Medline]
  30. Dieudonne MN, Pecquery R, Boumediene A, Leneveu MC, Giudicelli Y 1998 Androgen receptors in human preadipocytes and adipocytes: regional specificities and regulation by sex steroids. Am J Physiol 274:C1645–C1652
  31. Zang H, Ryden M, Wahlen K, Dahlman-Wright K, Arner P, Linden Hirschberg A 2007 Effects of testosterone and estrogen treatment on lipolysis signaling pathways in subcutaneous adipose tissue of postmenopausal women. Fertil Steril 88:100–106[CrossRef][Medline]
  32. Anderson LA, McTernan PG, Harte AL, Barnett AH, Kumar S 2002 The regulation of HSL and LPL expression by DHT and flutamide in human subcutaneous adipose tissue. Diabetes Obes Metab 4:209–213[CrossRef][Medline]
  33. Bluher S, Kratzsch J, Kiess W 2005 Insulin-like growth factor I, growth hormone and insulin in white adipose tissue. Best Pract Res Clin Endocrinol Metab 19:577–587[CrossRef][Medline]
  34. Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, MacDougald OA 2000 Inhibition of adipogenesis by Wnt signaling. Science 289:950–953[Abstract/Free Full Text]
  35. Yang Q, Graham TE, Mody N, Preitner F, Peroni OD, Zabolotny JM, Kotani K, Quadro L, Kahn BB 2005 Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 436:356–362[CrossRef][Medline]
  36. Graham TE, Yang Q, Bluher M, Hammarstedt A, Ciaraldi TP, Henry RR, Wason CJ, Oberbach A, Jansson PA, Smith U, Kahn BB 2006 Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. N Engl J Med 354:2552–2563[Abstract/Free Full Text]
  37. Eckardstein A, Wu FC 2003 Testosterone and atherosclerosis. Growth Horm IGF Res 13(Suppl A):S72–S84
  38. Grunfeld C, Kotler DP, Dobs A, Glesby M, Bhasin S 2006 Oxandrolone in the treatment of HIV-associated weight loss in men: a randomized, double-blind, placebo-controlled study. J Acquir Immune Defic Syndr 41:304–314[CrossRef][Medline]
  39. Szeplaki G, Varga L, Valentin S, Kleiber M, Karadi I, Romics L, Fust G, Farkas H 2005 Adverse effects of danazol prophylaxis on the lipid profiles of patients with hereditary angioedema. J Allergy Clin Immunol 115:864–869[CrossRef][Medline]
  40. Langer C, Gansz B, Goepfert C, Engel T, Uehara Y, von Dehn G, Jansen H, Assmann G, von Eckardstein A 2002 Testosterone up-regulates scavenger receptor BI and stimulates cholesterol efflux from macrophages. Biochem Biophys Res Commun 296:1051–1057[CrossRef][Medline]
  41. Goodwin B, Watson MA, Kim H, Miao J, Kemper JK, Kliewer SA 2003 Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-{alpha}. Mol Endocrinol 17:386–394[Abstract/Free Full Text]
  42. Pullinger CR, Eng C, Salen G, Shefer S, Batta AK, Erickson SK, Verhagen A, Rivera CR, Mulvihill SJ, Malloy MJ, Kane JP 2002 Human cholesterol 7{alpha}-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype. J Clin Invest 110:109–117[CrossRef][Medline]
  43. Wang S, Lai K, Moy FJ, Bhat A, Hartman HB, Evans MJ 2006 The nuclear hormone receptor farnesoid X receptor (FXR) is activated by androsterone. Endocrinology 147:4025–4033[Abstract/Free Full Text]
  44. De Pergola G, Holmang A, Svedberg J, Giorgino R, Bjorntorp P 1990 Testosterone treatment of ovariectomized rats: effects on lipolysis regulation in adipocytes. Acta Endocrinol (Copenh) 123:61–66[Abstract/Free Full Text]
  45. Giudicelli Y, Dieudonne MN, Lacasa D, Pasquier YN, Pecquery R 1993 Modulation by sex hormones of the membranous transducing system regulating fatty acid mobilization in adipose tissue. Prostaglandins Leukot Essent Fatty Acids 48:91–100[CrossRef][Medline]
  46. Singh R, Artaza JN, Taylor WE, Braga M, Yuan X, Gonzalez-Cadavid NF, Bhasin S 2006 Testosterone inhibits adipogenic differentiation in 3T3-L1 cells: nuclear translocation of androgen receptor complex with β-catenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors. Endocrinology 147:141–154[Abstract/Free Full Text]
  47. Singh R, Artaza JN, Taylor WE, Gonzalez-Cadavid NF, Bhasin S 2003 Androgens stimulate myogenic differentiation and inhibit adipogenesis in C3H 10T1/2 pluripotent cells through an androgen receptor-mediated pathway. Endocrinology 144:5081–5088[Abstract/Free Full Text]
  48. Fan W, Yanase T, Nomura M, Okabe T, Goto K, Sato T, Kawano H, Kato S, Nawata H 2005 Androgen receptor null male mice develop late-onset obesity caused by decreased energy expenditure and lipolytic activity but show normal insulin sensitivity with high adiponectin secretion. Diabetes 54:1000–1008[Abstract/Free Full Text]
  49. Kennell JA, MacDougald OA 2005 Wnt signaling inhibits adipogenesis through β-catenin-dependent and -independent mechanisms. J Biol Chem 280:24004–24010[Abstract/Free Full Text]
  50. Jiang XC, Moulin P, Quinet E, Goldberg IJ, Yacoub LK, Agellon LB, Compton D, Schnitzer-Polokoff R, Tall AR 1991 Mammalian adipose tissue and muscle are major sources of lipid transfer protein mRNA. J Biol Chem 266:4631–4639[Abstract/Free Full Text]
  51. Quinet E, Tall A, Ramakrishnan R, Rudel L 1991 Plasma lipid transfer protein as a determinant of the atherogenicity of monkey plasma lipoproteins. J Clin Invest 87:1559–1566[Medline]
  52. Zhou H, Li Z, Hojjati MR, Jang D, Beyer TP, Cao G, Tall AR, Jiang XC 2006 Adipose tissue-specific CETP expression in mice: impact on plasma lipoprotein metabolism. J Lipid Res 47:2011–2019[Abstract/Free Full Text]
  53. Harada LM, Amigo L, Cazita PM, Salerno AG, Rigotti AA, Quintao EC, Oliveira HC 2007 CETP expression enhances liver HDL-cholesteryl ester uptake but does not alter VLDL and biliary lipid secretion. Atherosclerosis 191:313–318[Medline]
  54. Gautier T, Masson D, Jong MC, Duverneuil L, Le Guern N, Deckert V, Pais de Barros JP, Dumont L, Bataille A, Zak Z, Jiang XC, Tall AR, Havekes LM, Lagrost L 2002 Apolipoprotein CI deficiency markedly augments plasma lipoprotein changes mediated by human cholesteryl ester transfer protein (CETP) in CETP transgenic/ApoCI-knocked out mice. J Biol Chem 277:31354–31363[Abstract/Free Full Text]
  55. Bamba V, Rader DJ 2007 Obesity and atherogenic dyslipidemia. Gastroenterology 132:2181–2190[CrossRef][Medline]
  56. Salter AM, Fong BS, Jimenez J, Rotstein L, Angel A 1987 Regional variation in high-density lipoprotein binding to human adipocyte plasma membranes of massively obese subjects. Eur J Clin Invest 17:16–22[Medline]
  57. Avramoglu RK, Basciano H, Adeli K 2006 Lipid and lipoprotein dysregulation in insulin resistant states. Clin Chim Acta 368:1–19[CrossRef][Medline]
  58. Ginsberg HN, Zhang YL, Hernandez-Ono A 2005 Regulation of plasma triglycerides in insulin resistance and diabetes. Arch Med Res 36:232–240[CrossRef][Medline]



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