in older men, hypogonadism (i.e., low circulating testosterone and/or low bioavailable testosterone) is associated with a range of deleterious effects, including loss of skeletal muscle mass and increased subcutaneous and visceral adiposity (48, 60). There is a stronger association between insulin resistance and accumulation of visceral than subcutaneous fat (2, 3, 21), and reductions in insulin sensitivity precede the development of type 2 diabetes and metabolic syndrome (10, 44). Moreover, an increase in visceral adiposity is associated with increased tissue inflammation, which, in and of itself, can produce low-grade systemic inflammation (23). Testosterone administration improves insulin sensitivity, fasting glucose, and HbA 1c levels (59) and is known to increase lean mass and decrease whole body and visceral adiposity in hypogonadal men (7, 9, 15, 18, 19, 28, 42, 55, 57). Interestingly, androgen receptor (AR) expression is higher in visceral than subcutaneous fat (53), and testosterone administration decreased lipid uptake more in retroperitoneal and omental fat than in subcutaneous regions (32), explaining the ability of testosterone to selectively prevent fat accumulation in the visceral depot (32, 38).

Previous explanations for reductions in fat mass after testosterone treatment include inhibition of adipocyte differentiation or a decrease in adipocyte size (51, 52, 54). Testosterone regulates adiposity via direct AR-mediated pathways, as evidenced by the AR knockout mouse, which exhibits reduced lipolysis (64) and elevated visceral adiposity (45), and by the ability of nonaromatizable androgens to prevent orchiectomy (ORX)-induced elevations in adiposity to a magnitude similar to that of testosterone (66). In addition, testosterone may indirectly regulate adiposity via estrogen receptor (ER) activation following the tissue-specific aromatization of testosterone, which occurs primarily in fat (5), with ∼85% of total serum estradiol (E 2 ) being derived from peripheral aromatization and the remaining ∼15% originating from gonadal aromatization (30, 40). However, debate persists regarding the role of aromatase in mediating the effects of testosterone on adiposity, with Finkelstein et al. (20) reporting that anastrozole (AN, a nonsteroidal aromatase inhibitor) ablated the lipolytic effects of administered testosterone in androgen-deficient men. In contrast, data from our laboratory indicate that AN co-administration does not inhibit the ability of supraphysiological testosterone to prevent ORX-induced elevations in visceral adiposity (4) in rodents, and others have reported that administration of AN alone does not alter body composition in men, perhaps because aromatase inhibition robustly increases serum testosterone via a luteinizing hormone- and follicle-stimulating hormone-mediated pathway (11, 31, 61).

Mechanistic examination of the interactions of testosterone administration with fat is limited. Xu and colleagues investigated the effects of androgen status on lipolytic regulation in male rats and reported that ORX decreased catecholamine-induced lipolysis and quantity of β-adrenoceptors and that testosterone administration ameliorated these deficits (63). Testosterone also induces upregulation of β-adrenoceptors, resulting in enhancement of the effectiveness of catecholamines on the β 2 -adrenoceptor-mediated lipolytic cascade in vitro (2, 22), and adenylate cyclase activity was enhanced by either testosterone or dihydrotestosterone (a nonaromatizable androgen) in a cell culture model (62). Additional in vitro research has determined that testosterone inhibits lipoprotein lipase (LPL) expression in adipocytes (8) and appears to inhibit adipocyte precursor differentiation (13, 17, 25). The aforementioned studies illustrate several pathways through which testosterone influences adipocyte growth. However, the influence of aromatase in mediating testosterone-induced alterations in adipocyte growth- and differentiation-related gene expressions in visceral fat remains largely underexplored.

The purpose of this study was to examine the effects of 4 wk of supraphysiological testosterone enanthate (TEST) administration with or without AN co-administration on retroperitoneal fat mass and molecular markers related to adipose tissue physiology in older gonad-intact and ORX rats. Given the prior literature, we hypothesized that TEST would prevent visceral fat accumulation in ORX rats by chronically altering lipogenic- and/or lipolytic-related gene expression in retroperitoneal fat and that AN co-administration would not alter these effects.

Values are means ± SE. P ≤ 0.05 was defined as the threshold of significance. One-way ANOVAs (for normally distributed data) were used to separately analyze dependent variables, and protected least significant difference post hoc analyses were performed for multiple comparisons among groups when appropriate. When main effects were observed, between-treatment independent t -tests were performed. Kruskal-Wallis and Mann-Whitney U -tests were performed when data were not normally distributed (Levene's statistic: P ≤ 0.10), and the Holm-Bonferroni correction was utilized to correct for potential type I error, which can occur when multiple comparisons are performed. In addition, Pearson correlations were performed for all dependent variables relative to serum testosterone and retroperitoneal fat pad mass. Data were analyzed with the SPSS 23.0.0 statistical software package (IBM, Chicago, IL).

The production of brown fat-like adipocytes within white adipocytes, referred to as “brite cells” (brown in white cells), the action of which is referred to as “briting” of adipose tissue, was investigated. Uncoupling protein (UCP-1), also known as thermogenin, is a mitochondrial transporter protein present in the inner mitochondrial membrane of brown and white adipose tissue that acts as a proton carrier activated by free fatty acids and can dissipate the proton gradient before it can be used to provide energy for oxidative phosphorylation. UCP-1 was also examined as part of the briting of the adipose tissue pathway. To determine the effects of chronic testosterone administration on androgen signaling and steroidogenesis, cytochrome P- 450ssc (Cyp11a1), 3β-hydroxysteroid dehydrogenase Δ 5 →Δ 4 -isomerase (3βHSD), and cytochrome P -450 aromatase (Cyp19a1) mRNAs were assayed.

Specifically, to test our hypotheses, SREBP-1 (a regulator of genes required for fatty acid metabolism and lipid production) and associated markers of proliferation and differentiation, peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα), mRNAs were examined. Moreover, the metabolic markers FASN (a multienzyme protein that catalyzes fatty acid synthesis), hormone-sensitive lipase (LIPE, a key enzyme in the regulation of lipid stores that is encoded by the LIPE gene), ACC (a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to malonyl-CoA), and LPL (the rate-limiting enzyme for the import of triglyceride-derived fatty acids by adipose tissue for storage) were interrogated.

After RNA isolation (see above), total RNA concentrations were analyzed using a spectrophotometer (Nanodrop Lite, Thermo Scientific), and 2 μg of retroperitoneal RNA were reverse-transcribed into cDNA for real-time PCR (RT-PCR) analyses using a commercial qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). RT-PCR was performed using gene-specific primers ( Table 1 ) and SYBR Green-based methods in a RT-PCR thermal cycler (Bio-Rad Laboratories, Hercules, CA). Primers were designed using primer designer software (Primer3Plus, Cambridge, MA), and melt curve analyses demonstrated that one PCR product was amplified per reaction. Fold-change values from SHAM rats were determined using the Livak method: 2 −ΔΔC T , with the assumption that primer binding efficiency was 100%, where 2 −ΔC T = housekeeping gene C T − gene of interest C T and 2 −ΔΔC T (or fold change) = 2 −ΔC T value of experimental treatment/2 −ΔC T average of SHAM. β 2 -Microglobulin was used as a housekeeping gene, given that it remained stable across all treatments.

Retroperitoneal fat homogenates obtained from cell lysis (see Tissue preparation ) were prepared for Western blotting using 4× Laemmli buffer at 1 μg/μl. Thereafter, 20 μl of prepared samples were loaded onto hand-casted 12% SDS-polyacrylamide gels (CBS Scientific, San Diego, CA) and subjected to electrophoresis (200 V at 75 min) using premade 1× SDS-PAGE running buffer (CBS Scientific). Proteins were transferred to polyvinylidene difluoride membranes (Whatman, Westran Clear Signal), which were blocked for 1 h at room temperature with 5% nonfat milk powder. Membranes were incubated overnight at 4°C in 5% bovine serum albumin with the following primary antibodies: rabbit anti-rat fatty acid synthase (FASN, 1:1,000 dilution; Cell Signaling Technology, Danvers, MA), rabbit anti-rat acetyl-CoA carboxylase (ACC, 1:1,000 dilution; Cell Signaling Technology), rabbit anti-rat sterol regulatory element-binding protein-1 (SREBP-1, 1:100 dilution; Abcam, Cambridge, MA), and rabbit anti-rat GAPDH (1:1,000 dilution; Genetex, Irvine, CA). On the following day, membranes were incubated with anti-rabbit IgG secondary antibodies (1:2,000 dilution; Cell Signaling Technology) at room temperature for 1 h. Thereafter, membrane development was performed using an enhanced chemiluminescence reagent (Amersham, Pittsburgh, PA), and band densitometry was performed using a digitized gel documentation system and associated densitometry software (UVP). Specifically, SREBP-1, FASN, and ACC protein content was investigated.

For protein analyses, ∼50 mg of the retroperitoneal fat pad were extracted using standard dissection techniques and placed in 500 μl of ice-cold cell lysis buffer [20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM Na-EDTA, 1 mM EGTA, 1% Triton, 20 mM sodium pyrophosphate, 25 mM sodium fluoride, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 , and 1 μg/ml leupeptin]. Samples were then homogenized via micropestle manipulation, insoluble proteins from RIPA homogenates were removed by centrifugation at 500 g for 5 min, and supernatants were assayed for total protein content using a bicinchoninic acid protein assay kit (Thermo Scientific, Waltham, MA) prior to immunoblotting (see below). An additional 50 mg of the retroperitoneal fat pad were extracted and placed in 500 μl of RiboZol (Ameresco) for RNA isolation. Samples were then homogenized via micropestle manipulation, and RNA was isolated according to the manufacturer's recommendations. Unprocessed retroperitoneal fat pads removed during dissections were flash-frozen in liquid nitrogen and stored at −80°C for later potential analyses.

Adipose tissue samples were fixed in 10% formalin for ∼1 wk and washed in cold running tap water. The segments were placed into embedding cassettes and immediately stored in 70% alcohol for processing. The tissue-processing procedure included the following steps: dehydration, clearing, and infiltration. Dehydration was accomplished by gradual increases in percentages of ethyl alcohol, and tissue was subsequently embedded in paraffin. Paraffin tissue blocks were sectioned into 6-μm slices and then placed onto glass microscope slides. Paraffin was removed with xylene, and sections were stained with hematoxylin and eosin to visualize adipocytes. Microscope images of stained slides were photographed with a ×40 objective using a digital camera and associated software (Nikon Eclipse Ti-U). Ten images per sample were obtained for quantification purposes. Adipocyte cross-sectional areas from digitally saved images were quantified using ImageJ (National Institutes of Health, Bethesda, MD).

All serum samples were assayed in duplicate within the same run using commercial kits. Testosterone was determined via enzyme-linked immunosorbent assay (EIA; ALPCO Diagnostics, Salem, NH) with a sensitivity of 0.022 ng/ml and an intra-assay covariance of 7.97%. E 2 was determined by EIA (ALPCO Diagnostics) with a sensitivity of 1.399 pg/ml and an intra-assay covariance of 6.35%. In addition, the sensitivity of the E 2 EIA was increased by serial dilution of the lowest nonzero standard, as previously described by our laboratory ( 65 ). Nonesterified fatty acids (NEFA) were determined by EIA (Wako Diagnostics, Mountain View, CA) with a sensitivity estimated to be 0.0014 meq/l and an intra-assay covariance of 4.91%.

The dose and route of TEST were chosen to mimic a recent clinical trial that was conducted in our laboratory to investigate the musculoskeletal and body composition effects of higher-than-replacement TEST administration in elderly hypogonadal men ( 9 ). This administration regimen elevates circulating testosterone to the supraphysiological range for ∼7 days in rats ( 67 ), which increases the likelihood of systemic aromatization. In addition, this dosing regimen has consistently been shown to prevent cancellous bone loss and produce potent myotrophic effects in young and skeletally mature rodents following ORX ( 35 , 66 , 67 ), while lower doses do not provide full musculoskeletal protection. AN was administered in a dose exceeding that which produced a marked reduction in circulating E 2 and dramatically elevated circulating testosterone (a hallmark characteristic of aromatase inhibition) in intact female rodents within 7 days of treatment ( 47 ). Pumps were refilled biweekly to ensure that AN was available continuously throughout the study.

On day 42 , representing 29 days after initiation of TEST/AN treatment and 24 h after the last TEST injection, rats were euthanized with pentobarbital (120 mg/kg ip), blood was collected via cardiac puncture, and serum aliquots were stored at −80°C. The retroperitoneal fat pad, prostate, and levator ani-bulbocavernosus muscle complex (LABC) were harvested, weighed, snap-frozen in liquid nitrogen, and stored at −80°C for analyses. We evaluated these tissues, because the retroperitoneal fat pad is a viscerally located adipose tissue that is particularly sensitive to the effects of ORX and TEST administration in young and mature rodents, while the prostate and LABC nonadipose androgen-sensitive tissues are commonly used to demonstrate the effectiveness of androgen treatment ( 35 , 66 – 68 ).

For the present study, we examined a random subset of retroperitoneal adipose tissue from our previously published companion articles ( 4 , 37 ). Briefly, tissue was acquired from rats ( n = 6/group) that were stratified by initial weight and divided into the following groups: 1 ) sham-operated (SHAM), 2 ) ORX, 3 ) ORX + TEST (7.0 mg·wk −1 ·animal −1 ), and 4 ) ORX + TEST + AN (0.5 mg·day −1 ·animal −1 ). Animals were subjected to aseptic bilateral closed ORX (removal of testes, epididymis, and epididymal fat) or SHAM surgery performed by Charles River Laboratories. After ORX or SHAM surgery, rats received no intervention for 2 wk to ensure that they fully recovered from surgery and that the influence of endogenous gonad-derived sex steroid hormones had subsided. Subsequently, animals received AN (Sigma-Aldrich, St. Louis, MO) or vehicle (50% DMSO, 45% saline, and 5% ethanol b.v.) infused at a continuous rate of 2 μl/h via subcutaneously implanted computer-programmed iPRECIO microinfusion drug pumps (Primetech, Tokyo, Japan) and TEST (Savient Pharmaceutical, East Brunswick, NJ) or vehicle (sesame oil) once weekly via intramuscular injection under brief isoflurane administration. Five intramuscular injections were administered over a 29-day testing period, on postsurgery days 14, 21, 28, 35 , and 41 . Serum samples were collected prior to each intramuscular injection, and aliquots were stored at −80°C for analyses.

Barrier-raised and specific pathogen-free 10-mo-old male Fischer 344 rats (Charles River Laboratories, Wilmington, MA) were individually housed in a temperature- and light-controlled room on a 12:12-h light-dark cycle. The animals were fed rodent chow containing 3.1 kcal/g, distributed as 58% carbohydrate, 24% protein, and 18% fat, with 1.0% calcium and 0.7% phosphorus (2018 Teklad Global 18% Protein Rodent Diet, Harlan Laboratories, Indianapolis, IN), and tap water ad libitum. All experimental procedures conformed to the Institute of Laboratory Animal Resources Guide to the Care and Use of Experimental Animals and were approved by the Institutional Animal Care and Use Committee at the Malcom Randall Veterans Affairs Medical Center.

Within the entire cohort, FASN mRNA expression was negatively associated with serum testosterone ( r = −0.499, P = 0.013; Fig. 9 ) and positively associated with retroperitoneal fat mass ( r = 0.534, P = 0.007). LPL mRNA expression was negatively associated with serum testosterone ( r = 0.508, P = 0.011; Fig. 9 ) and positively associated with retroperitoneal fat mass ( r = −0.439, P = 0.032). Relationships among SREBP-1 mRNA expression and serum testosterone ( P = 0.077) or relative retroperitoneal fat mass ( P = 0.058) approached significance and are presented in Fig. 9 . No significant relationships were observed between serum E 2 and FASN mRNA ( r = 0.045, P = 0.836), LPL mRNA ( r = 0.035, P = 0.872), or SREBP-1 mRNA ( r = −0.128, P = 0.553) or between LIPE mRNA and serum testosterone, serum E 2 , or retroperitoneal fat mass ( r = −0.053, P = 0.805; r = −0.145, P = 0.499; and r = 0.296, P = 0.161, respectively; data not shown).

Group differences for ACC protein are presented in Fig. 7 (ANOVA: P = 0.049). ACC protein expression was not different among SHAM and ORX animals ( P = 0.576) but was 102% and 82% higher in ORX + TEST ( P = 0.016) and ORX + TEST + AN ( P = 0.057, trend), respectively, than SHAM animals. ACC protein was also 80% higher in ORX + TEST than ORX animals ( P = 0.042). No differences were observed among groups for SREBP-1 ( P = 0.571) or FASN ( P = 0.577) protein expression.

Significant differences for LPL mRNA are presented in Fig. 5 (ANOVA: P = 0.001). LPL mRNA was 84% higher in ORX than SHAM animals ( P = 0.001). LPL mRNA was 95% lower in ORX + TEST ( P < 0.001) and 47% lower in ORX + TEST + AN ( P = 0.043) than ORX animals. In addition, LPL mRNA was 48% lower in ORX + TEST than ORX + TEST + AN animals ( P = 0.042). There were no differences in LPL mRNA among the ORX + TEST or ORX + TEST + AN and SHAM animals ( P = 0.600 and P = 0.117, respectively).

Significant group differences for FASN mRNA (ANOVA: P = 0.006) are presented in Fig. 5 . FASN mRNA was 2.5-fold higher after ORX than SHAM ( P = 0.012) and lower in ORX + TEST than ORX ( P = 0.001) and ORX + TEST + AN ( P = 0.050) animals. A trend also suggested that FASN mRNA was lower in ORX + TEST + AN than ORX animals ( P = 0.061). No differences were present among SHAM and ORX + TEST ( P = 0.189) or ORX + TEST + AN ( P = 0.450) animals.

Fig. 4. Longitudinal effects of TEST + AN on serum concentrations of nonesterified fatty acids (NEFA). Statistically significant absolute values are represented as percent change from baseline. P values are from within-group repeated-measures ANOVA and LSD post hoc analysis of between-group and between-time point differences in absolute values. Percent changes in serum NEFA are shown for group and time point. Sac, euthanasia. Values are means ± SE. a–d P ≤ 0.05 vs. respectively labeled group: SHAM (a), ORX (b), ORX + TEST (c), and ORX + TEST + AN (d). * P ≤ 0.01.

Repeated-measures analyses of NEFA revealed significant group and time point differences (ANOVA: P < 0.001; Fig. 4 ). NEFA were higher in ORX + TEST than ORX animals at day 7 ( P = 0.019), day 14 ( P = 0.013), and day 21 ( P = 0.018), but not day 28 . NEFA increased in ORX + TEST animals at day 7 of testosterone administration from 0.513 to 0.625 meq/l ( P = 0.004) and remained elevated above baseline at day 14 (0.589 meq/l, P = 0.028). Serum NEFA levels were higher in ORX + TEST than SHAM (0.414 meq/l, P = 0.019) and ORX + TEST + AN (0.501 meq/l, P = 0.008) animals at day 7 . At day 21 , serum NEFA concentrations were higher in ORX + TEST than ORX + TEST + AN animals ( P = 0.042). Upon euthanization, serum NEFA was higher in ORX + TEST (0.495 meq/l) than ORX + TEST + AN (0.379 meq/l) animals only ( P = 0.036). There were no differences in serum NEFA levels between groups at baseline ( P = 0.689), and no significant changes were observed in SHAM animals at any time point ( P > 0.05).

Group differences for serum testosterone and E 2 (ANOVA: P < 0.001 for testosterone and P = 0.042 for E 2 ) and for the testosterone-to-E 2 ratio (ANOVA: P < 0.001, on a pg/ml basis) are presented in Fig. 3 . Serum testosterone was ∼97% lower after ORX than SHAM ( P = 0.004) and ∼415% and 380% higher in ORX + TEST and ORX + TEST + AN, respectively, than SHAM ( P < 0.001) animals, with no differences between ORX + TEST and ORX + TEST + AN animals ( P = 0.547). Serum E 2 was increased 62% in ORX + TEST compared with SHAM animals ( P = 0.024), whereas E 2 in ORX + TEST + AN animals was not different from SHAM animals ( P = 0.784). The testosterone-to-E 2 ratio was lower in ORX than SHAM ( P = 0.048) and higher in ORX + TEST than ORX ( P = 0.007) and SHAM ( P = 0.022) animals. The testosterone-to-E 2 ratio was also higher in ORX + TEST + AN than ORX ( P < 0.001), SHAM ( P = < 0.001), and ORX + TEST ( P = 0.031) animals.

We previously reported the prostate and LABC mass and serum testosterone concentrations for the entire cohort ( 4 ). Values reported here are for the subset ( n = 6) analyzed in this subanalysis and are necessary to characterize the magnitude of the effects reported. Group differences for LABC and prostate mass are presented in Fig. 3 (ANOVA: P < 0.001 for both measures). LABC mass was 43% lower in ORX than SHAM animals ( P = 0.002). LABC mass was 44% and 49% higher in ORX + TEST and ORX + TEST + AN, respectively, than SHAM animals ( P < 0.01). In addition, LABC mass was 154% and 163% higher in ORX + TEST and ORX + TEST + AN, respectively, than ORX animals ( P < 0.001). Prostate mass was 64% lower in ORX than SHAM animals ( P = 0.0087). Prostate mass was 59% and 76% higher in ORX + TEST and ORX + TEST + AN, respectively, than SHAM animals ( P < 0.05). In addition, prostate mass was ∼350% and 400% higher in ORX + TEST and ORX + TEST + AN, respectively, than ORX animals ( P < 0.001). There were no differences in LABC mass ( P = 0.788) or prostate mass ( P = 0.512) between ORX + TEST and ORX + TEST + AN animals. A significant main effect was also observed among treatments for the LABC mass-to-prostate mass ratio (ANOVA: P = 0.023), which illustrates the anabolic-to-androgenic ratio of androgens ( 66 ) and the sensitivity of specific tissues to androgen status. This ratio was 78% higher after ORX than SHAM (trend, P = 0.089) and 51% and 55% lower in ORX + TEST and ORX + TEST + AN, respectively, than ORX animals ( P < 0.05). No differences in the LABC mass-to-prostate mass ratio were present among the SHAM, ORX + TEST, or ORX + TEST + AN ( P = 0.401 and P = 0.536, respectively) or among the ORX + TEST and ORX + TEST + AN ( P = 0.313) animals.

Fig. 1. Effects of testosterone enanthate (TEST) + anastrozole (AN) on body mass and retroperitoneal fat mass. Fisher 344 rats ( n = 6/groups) were subjected to sham surgery (SHAM) or orchiectomy (ORX). Androgen-treated groups received TEST (7.0 mg/wk, ORX + TEST) or TEST + AN (0.5 mg/day, ORX + TEST + AN). A : retroperitoneal fat mass normalized to body mass at euthanization (Sac). Pre-Sx, before group stratification. Values are means ± SE reported from ANOVA and least significant difference (LSD) post hoc analyses. a–d Significant ( P ≤ 0.05) within-group differences at each time point compared with initial values; a, SHAM; b, ORX; c, ORX + TEST; and d, ORX + TEST + AN. * P ≤ 0.01, ** P ≤ 0.001. 1,2 Significant differences between androgen-treated (ORX + TEST and ORX + TEST + AN) and SHAM animals at each time point ( P ≤ 0.05). B : change in body mass relative to initial body mass before surgical implantation of the micropump. C : nonnormalized retroperitoneal fat mass at euthanization. D : body mass prior to group stratification. E : body mass at euthanization (Sac). Values are means ± SE reported from ANOVA and LSD post hoc analyses. a–d Significantly different ( P ≤ 0.05) from respectively labeled groups: SHAM (a), ORX (b), ORX + TEST (c), and ORX + TEST + AN (d). * P ≤ 0.01, ** P ≤ 0.001.

Group differences for body mass change and retroperitoneal fat mass (ANOVA: P < 0.001) are presented in Fig. 1 . No differences were present among groups for presurgical body mass ( P = 0.099) or for body mass at euthanization ( P = 0.191). However, differences among groups were observed for change in body mass relative to initial body mass ( P = 0.038), with the ORX + TEST + AN animals gaining the most body mass. Because of the differences in body mass change among groups, we report both absolute and relative (normalized to body mass) fat mass; however, both methods of evaluation produced similar outcomes. Briefly, absolute and relative retroperitoneal fat masses were 29/34% (absolute/relative) higher after ORX than SHAM ( P < 0.001). Both TEST treatments prevented the ORX-induced increase in retroperitoneal fat: absolute/relative fat mass was 61/64% lower in ORX + TEST ( P < 0.001) and 38/39% lower in ORX + TEST + AN ( P < 0.001) than ORX animals. Absolute/relative fat mass was also 20/27% lower in ORX + TEST than SHAM animals (absolute: P < 0.001, relative: P = 0.001) and 19/20% lower than ORX + TEST + AN animals (absolute: P = 0.007, relative: P = 0.012). There were no differences in absolute ( P = 0.177) or relative ( P = 0.358) retroperitoneal fat mass among the SHAM and ORX + TEST + AN animals. There were no differences in adipocyte cross-sectional area ( P = 0.474), adipocyte number ( P = 0.868), or adipocyte cross-sectional area-to-adipocyte number ratio ( P = 0.614) between any treatment groups ( Fig. 2 ).

DISCUSSION

Our understanding of adipose tissue as a dynamic endocrine organ, and not merely a reservoir for energy storage, continues to advance. Indeed, it is known that adipose tissues express and secrete a diverse range of bioactive peptides, known as adipokines, that act at local and systemic levels, exerting their effects on neuroendocrine and immune function and energy metabolism (29). Adipose tissue excess, especially in the visceral compartment, is strongly associated with insulin resistance (2, 3, 21), development of type 2 diabetes and metabolic syndrome, and an increased risk of cardiovascular disease (10, 29, 41, 44). Hypogonadism is associated with loss of skeletal muscle mass and increased visceral adiposity (48, 60) in older men, and a growing body of evidence supports an inverse relationship between the degree of testosterone deficiency and the severity of coronary artery disease (41). Interestingly, testosterone administration is known to improve subcutaneous and visceral adiposity and the associated morbidities known as metabolic syndrome (7, 9, 15, 18, 19, 28, 42, 55, 57, 58), exerting its effects on fat directly via AR binding and indirectly after tissue-specific aromatase enzyme conversion of testosterone to E 2 and ER binding (4).

In the present study we examined retroperitoneal fat mass and lipolytic/lipogenic-related gene and protein expression in visceral adipose tissue in 10-mo-old ORX rats, where serum testosterone (i.e., the sole androgenic substrate for aromatase) was essentially ablated, and in ORX rats after chronic TEST treatment without or with AN (a nonsteroidal aromatase inhibitor). Collectively, these treatments resulted in a stepwise increase in the testosterone-to-E 2 ratio, with ORX exhibiting subphysiological serum testosterone and normal E 2 (i.e., the lowest testosterone-to-E 2 ratio), SHAM animals exhibiting normal testosterone and normal E 2 , ORX + TEST animals exhibiting supraphysiological testosterone and high E 2 , and ORX + TEST + AN animals exhibiting supraphysiological testosterone with normal E 2 (i.e., the highest testosterone-to-E 2 ratio). The primary findings of this study were that aromatase action 1) mediated the chronic antilipogenic effects of supraphysiological testosterone in visceral fat and 2) is required to complete the androgen-induced visceral fat loss after ORX. Specifically, TEST + AN completely prevented the ORX-induced increase in retroperitoneal fat mass, while TEST alone reduced visceral fat mass to below that of gonad-intact SHAM and ORX + TEST + AN animals, indicating that aromatase activity and/or elevated circulating E 2 enhance, but are not required for, testosterone-mediated reductions in visceral adiposity.

We previously reported that trenbolone enanthate (a nonaromatizeable synthetic testosterone analog) potently reduced visceral fat mass in young and older ORX animals, indicating that fat loss occurs in response to androgen administration, even in the absence of an androgenic substrate for aromatase (4, 35, 66). However, our previous work did not account for the possibility that androstendione (derived from dehydroepiandrosterone) can be aromatized to estrone and, subsequently, converted to E 2 by actions of 17β-hydroxysteroid dehydrogenase in tissues, such as fat, expressing the required enzymes (50). Our current work addresses this with the use of AN, a potent nonsteroidal aromatase inhibitor, to pharmacologically inhibit both the traditional testosterone- and alternative androstendione-mediated pathways of E 2 synthesis and demonstrates that aromatase activity enhances, but is not required for, androgen-induced fat loss. The strong negative association between serum testosterone and retroperitoneal fat mass and the lack of an association between visceral fat mass and circulating E 2 in our animals further support the contention that testosterone is able to directly stimulate fat loss, at least when serum testosterone is elevated to the supraphysiological range and when E 2 remains in the normal physiological range; however, future work evaluating adipose-specific sex steroid hormone concentrations would further support this contention, given that fat is the primary site of aromatase activity in males (5). Nevertheless, our findings appear to support those of Finkelstein et al., who reported that androgen-deficient men receiving testosterone treatment with or without AN exhibited fat loss that was at least partially mediated via aromatase (20).

Androgen-mediated fat loss is typically described as lipolytic and is partially mediated via LIPE (14, 34), which hydrolyzes stored triacylglycerols in adipose tissue. Here, we report that LIPE mRNA was higher in ORX than ORX + TEST animals and that aromatase inhibition prevented the testosterone-induced reduction in LIPE mRNA in visceral adipose tissue, indicating that LIPE mRNA is chronically elevated in the absence of testosterone (i.e., the androgenic substrate for aromatase) and when aromatase activity is pharmacologically inhibited. These results are difficult to explain, given that others have reported that testosterone stimulates LIPE activity directly through androgen receptor-mediated pathways and indirectly via increases in circulating catecholamines (14). On the basis of these results, it appears that testosterone may initially upregulate genes responsible for increased lipolysis, while the chronic androgen-induced maintenance of visceral adiposity may result from other mechanisms. Indeed, in the current study, circulating NEFA were initially increased in ORX + TEST animals only and then returned to SHAM levels. Furthermore, the reduced serum NEFA in ORX and ORX + TEST + AN compared with ORX + TEST animals indicates that aromatase activity at least partially mediates the ability of testosterone to reduce and maintain visceral fat mass.

Lipogenesis is the process by which fatty acids and triglycerides are synthesized in the liver or adipose tissue, and adipogenesis refers to the differentiation of preadipocytes into mature fat cells (56). Some evidence indicates that androgens reduce fat mass by directing mesenchymal pluripotent stem cells toward the myogenic and away from the adipogenic lineage (52). Interestingly, the results of the present study also demonstrate a downregulation of key genes involved in regulation of fatty acid synthesis in visceral fat of ORX + TEST animals, but not in those undergoing aromatase inhibition. In this regard, SREBP-1, which is expressed in the liver and adipose tissue, is the main transcription factor of the lipogenic pathway (49, 56) and is linked to lipid synthesis (27). We observed that SREBP-1 mRNA was increased after ORX and that ORX + TEST prevented this increase, while SREBP-1 mRNA remained elevated in ORX + TEST + AN animals, indicating that SREBP-1 mRNA is elevated in visceral fat by the absence of testosterone (i.e., the androgenic substrate for aromatase) and when aromatase activity is inhibited pharmacologically via AN. In support of this concept, serum E 2 was highest in ORX + TEST animals, which exhibited the lowest SREBP-1 mRNA. However, no differences in SREBP-1 protein expression were present among groups at the chronic time point evaluated in this study, and no relationship was observed between serum E 2 and SREBP-1 mRNA. SREBP-1 also exerts proadipogenic effects by inducing PPARγ (46), a regulator of adipogenesis that is required for expression of the adipocyte phenotype in precursor cells (27, 46). However, we did not observe changes in PPARγ or C/EBPα mRNA, a principal transcription factor that maintains the adipocyte-differentiated state (39, 46).

LPL is the main enzymatic regulator of triglyceride uptake by adipocytes that acts by hydrolyzing circulating triglyceride-filled lipoproteins and initiating adipocyte free fatty acid uptake and triglyceride accumulation. Hypogonadism increases LPL activity in men (24), and testosterone treatment reduces LPL activity and triglyceride uptake in subcutaneous abdominal adipose tissue in hypogonadal men (33). In the present study, ORX increased LPL mRNA expression, likely contributing to the large increase in visceral fat accumulation, and both ORX + TEST and ORX + TEST + AN animals maintained LPL mRNA at SHAM levels, which supports previous literature indicating that testosterone inhibits lipid uptake by negatively regulating LPL (12, 16). However, LPL mRNA was higher in ORX + TEST + AN than ORX + TEST animals, indicating that aromatase activity at least partially mediates the suppressive effects of testosterone on LPL. Interestingly, the androgen-induced suppression of LPL appears to occur more prevalently in visceral than subcutaneous adipose tissue after testosterone administration (32), likely because androgens differentially regulate lipogenic gene expression based on localization and/or AR expression (53).

ACC and FASN are lipogenic enzymes that irreversibly carboxylate acetyl-CoA to malonyl-CoA and convert malonyl-CoA to fatty acids for storage as triglycerides, respectively, and are linked to visceral fat accumulation (6). In the present study, retroperitoneal fat ACC mRNA and protein expression were similar among SHAM and ORX groups, while both testosterone-treated groups exhibited elevated ACC protein expression compared with the SHAM group. In contrast, ORX increased FASN mRNA expression, likely contributing to the retroperitoneal fat accumulation in this group, and both testosterone treatments prevented this effect, which may have contributed to fat loss. Interestingly, the reduction in FASN mRNA was greater in ORX + TEST than ORX + TEST + AN animals, suggesting that testosterone and E 2 coregulate FASN expression, which is required for conversion of malonyl-CoA to fatty acids. In support of this concept, serum testosterone, but not E 2 , was associated with reduced lipogenic (FASN and LPL) mRNA expression across groups; however, no differences in visceral fat FASN protein were present among groups. Our findings parallel investigations reporting lower FASN mRNA expression in visceral adipose tissue of lean than obese humans, despite similar levels of FASN protein expression among the lean and obese individuals (36).

Collectively, our results indicate that testosterone and aromatase activity coregulate lipogenic gene expression in visceral adipose tissue, as evidenced by the greater suppression of FASN and LPL mRNA in retroperitoneal fat of ORX + TEST than ORX + TEST + AN animals and by the ability of AN co-administration to ablate the testosterone-induced suppression of SREBP-1 mRNA in visceral fat. Indeed, FASN and LPL mRNA expression were negatively associated with testosterone and positively associated with retroperitoneal fat mass, suggesting that reduced lipogenesis mediates the reduction in visceral adiposity in response to chronic testosterone administration. In addition, the concomitant increase in LIPE and ACC mRNA expression in ORX + TEST + AN animals suggests that aromatase inhibition produces a combined stimulation of lipogenic/lipolytic pathways that may inhibit cellular transport of fatty acids available for β-oxidation, which could underlie the higher visceral fat mass in ORX + TEST + AN than ORX + TEST animals. Indeed, ACC regulates the metabolism of fatty acids through irreversible carboxylation of acetyl-CoA to malonyl-CoA, which directly inhibits carnitine-palmitoyl transferase (CPT I) and prevents importation of newly synthesized fats into mitochondria and rapid oxidation of these newly synthesized fats (1).

To more fully explore the mechanisms of androgen-mediated fat loss, we examined pathways involving PR domain-containing-16 (PRDM16)-induced briting of white adipose tissue. PRDM16 is an adipose tissue transcription coregulatory protein that stimulates production of brown fat-like adipocytes within white adipose tissue (i.e., briting, or beige adipocytes), the presence of which leads to a significant upregulation of brown adipose tissue-selective genes, including UCP-1 and Cidea, an important regulator of lipolysis. Previous work indicates that exposure of female mouse pups to testosterone downregulated UCP-1 and Cidea mRNA in brown adipose tissue in adulthood (26). In the current study, PRDM16 mRNA was not altered in visceral (white) fat at the chronic time point we evaluated, while UCP-1 mRNA was reduced by ORX and TEST did not prevent this effect. As such, we find it unlikely that the briting of white adipose tissue contributed to testosterone-induced fat loss; however, a more thorough time-course evaluation of the transcriptomic and protein expression responses of visceral fat is required to fully exclude this possibility. In contrast, Cidea mRNA was reduced in ORX + TEST + AN compared with SHAM and ORX animals, which may have contributed to the reduced retroperitoneal fat mass in this group, given that mice lacking a functional Cidea exhibit higher metabolic rates and higher lipolysis in brown adipose tissue and that depletion of Cidea markedly elevates lipolysis in human adipocytes (43).