Introduction

Obesity is associated with an increased risk to develop the metabolic syndrome, type 2 diabetes, and cardiovascular disease (1). Sex steroids are important regulators of fat metabolism in men. Androgens may regulate adipose tissue metabolism in men either directly by stimulation of the androgen receptor (AR)¹ or indirectly by aromatization of androgens into estrogens and, thereafter, by stimulation of the estrogen receptors (ERs). Clinical case reports have documented disturbances of adipose tissue metabolism including obesity in aromatase-deficient and estrogen-resistant men (2, 3, 4, 5, 6), and studies using ER-inactivated male mice have shown that ER- but not ER-inactivated mice are obese (7, 8), showing that ER activation results in reduced fat mass in men. Because previous studies, investigating the effect of androgens on adipose tissue metabolism, have used aromatizable androgens, resulting in a mixture of ER and AR activation, the role of specific AR activation for the regulation of adipose tissue metabolism in men is largely unknown. Previous studies have shown that testosterone treatment reduces the amount of visceral fat in middle-aged men (9). Recently it was shown that AR-inactivated male mice develop late-onset obesity, but because serum testosterone levels were drastically decreased because of atrophic testes in these mice, it was impossible to exclude the possibility that the effect on fat mass in these mice simply reflected the impaired action of converted estrogen from serum testosterone on adipose tissue (10, 11). A loss of estrogenic action as the cause of the obesity in these mice was supported by the finding that the fat mass was reduced by estrogen treatment. Women with polycystic ovary syndrome are hyperandrogenic, and 50% of women with polycystic ovary syndrome are obese with an increase in the visceral adipose tissue, suggesting that elevated androgens might increase the amount of fat mass in women (12). In conclusion, all these findings are consistent with the notion that ER activation results in reduced fat mass. In contrast, the interpretations of the effect of AR activation, or rather mixed AR/ER activation, on fat mass are conflicting. To directly compare in vivo in men the effect of AR activation on fat mass with the effect of ER activation, orchidectomized (orx) mice were treated with the non-aromatizable androgen 5-dihydrotestosterone (DHT) or 17-estradiol (E2). We show that AR activation results in obesity and altered lipid metabolism in orx mice.

Research Methods and Procedures

Animals had free access to fresh water and soy-free food pellets (R70; Lactamin AB, Stockholm, Sweden or 2016; Harlan Teklad, Oxon, United Kingdom). For the long-term experiment, 3-month-old male mice were either sham-operated or orx and treated for 5 weeks with DHT (45 g/d) or E2 (0.05 g/d) administered through subcutaneous silastic implants (Silclear Tubing; Degania Silicone, Jordan Valley, Israel) in the cervical region (13). For the testosterone and aromatase inhibitor experiment, 23-day-old male mice (C57Bl6/N) were either sham-operated or orx and treated for 5 weeks with testosterone (1.5 g/d) or testosterone plus aromatase inhibitor (anastrazole, Arimidex; 250 g/d; AstraZeneca, London, UK). Testosterone (Serva Electrophoresis GmbH, Heidelberg, Germany) was administered through subcutaneous silastic implants. Anastrazole is a potent, non-steroidal, highly selective aromatase inhibitor with no intrinsic hormonal activity (14). The substance was obtained from AstraZeneca after formal approval of the protocol and was administered orally. For elucidating the effects of DHT on fat mass in the absence of ER, 9-month-old ER^{- /-} male mice were either sham-operated or orx and treated with DHT (45 g/d) or E2 (0.05 g/d) for 4 weeks (15). The ER^{- /-} mice were of a mixed C57BL/6J/129 background (16). For the short-term experiment, 3-month-old male mice were orx and treated with DHT (45 g/d) for 1 week before measuring VO₂, VCO₂, resting metabolic rate, locomotor activity, and food consumption and collection of urine and feces. Blood samples were taken from freely fed animals. Vehicle (V) animals received empty implants. E2 and DHT were obtained from Sigma Chemical Co. (St. Louis, MO). All animal procedures were approved by the local ethics committee on animal care at Göteborg University or at Katholieke Universiteit Leuven and were conducted in accordance with guidelines.

Indirect Calorimetry

Mice were housed in separate chambers, and the volume of O₂ consumed (VO₂) and the volume of CO₂ produced (VCO₂) were measured by indirect calorimetry by an Oxymax system (Columbus Instruments, Columbus, OH) for 120 minutes at 30 °C (thermoneutrality). A steady-state period was reached after 90 minutes and the following 30 minutes between 90 and 120 minutes were used for statistical calculations. VCO₂ and VO₂ were measured every sixth minute and are presented as a mean of every second measurement. Respiratory quotient (RQ) was measured as the ratio of VCO₂ to VO₂. Energy expenditure (EE; kilocalories per minute per kilogram^0.75) was calculated using a rearrangement of the Weir equation as supplied by Columbus Instruments: (3.815 + 1.232 RQ) VO₂. Fat and glucose oxidation (grams per minute per kilogram^0.75) were calculated using the following formulas (17): (1.689 VO₂) - (1.689 VCO₂) and (4.12 VCO₂) - (2.91 VO₂), respectively.

Activity Measurement

With the mice in separate chambers, the spontaneous physical activity was measured simultaneously using a photocell-based activity monitor (Opto-Max; Columbus Instruments). Interruption of four infrared photocells on the short side and eight photocells on the long side of the cage recorded the activity. Broken beams were recorded every 30 seconds for 2 hours.

Food Consumption and Urine and Feces Collection

One week after orx and start of DHT treatment, mice were kept individually in metabolic cages (locally designed urine collection cages; G. Bergström, Department of Physiology, Göteborg, Sweden) for 5 days. Throughout the experiments, the mice had free access to chow and drinking water. Water and food intake, feces output, urine volume, and body weight were measured every 24 hours, and the 3 last days were used for analyses.

Lipoprotein Size Distribution

The size distribution profiles of lipoproteins were measured using a high performance liquid chromatography system, SMART, as previously described (18). In brief, 10 L of serum was loaded on a Superose 6 PC 3.2/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden), and the chromatographic system was linked to an air segmented continuous flow system for on-line post-derivatization analysis of total cholesterol. The SMART system was connected to a sample injector (Gina 50, Gynkotek HPLC; Gynkotek GmbH, Germering, Germany), and the elution buffer (0.01 M Tris, 0.03 M NaCl, pH 7.4) had a flow rate of 35 L/min. The integrated area of the fractions was expressed in molar concentration.

DNA Microarray Analysis

mRNA from the livers from the long-term experiment (n = 5 for each treatment group) was prepared using RNeasy Mini Kit including an on-column DNase digestion step using the RNase-free DNase set (Qiagen, Chatsworth, CA). The mRNA samples derived from each individual mouse were reverse transcribed into cDNA, labeled, and analyzed using DNA microarray (mouse expression set 430; Affymetrix, Santa Clara, CA; n = 5 for each treatment group). Preparation of labeled cRNA and hybridization was done according to the Affymetrix Gene Chip expression analysis manual. Scanned output files were analyzed using Affymetrix Micro Array Suite Version 5.0 software. To allow comparison of gene expression, chips were globally scaled to an average intensity of 500. We exclusively studied the expression profile of genes known to be of importance for the regulation of lipoprotein metabolism, including the biosynthesis of cholesterol, fatty acids, and triglycerides. The absolute signals generated were used for analyses, and ANOVA followed by Dunnett's multiple comparison test was used as the statistical analysis method.

Real-time Polymerase Chain Reaction Analysis

mRNA from the brown adipose tissue was prepared from the long-term experiment (n = 4–6) using RNeasy Lipid Tissue Mini Kit including an on-column DNase digestion step using the RNase-free DNase set (Qiagen). cDNA was synthesized and amplified using the ABI Prism 7000 Sequence Detection System (PE Applied Biosystems, Stockholm, Sweden). Predesigned primers and probes for uncoupling protein 1 (Mm00494069_m1) and carnitine palmitoyltransferase-1 (CPT-1) (Mm00487200_m1) were used, and 18S rRNA was used as an internal standard. The mRNA amount of each gene was calculated using the "standard curve method" and adjusted for the expression of 18S rRNA.

Results

DHT Treatment Increases Body Weight and Fat Mass in orx Male Mice

To directly compare in vivo in male mice the effect of AR activation with the effect of ER activation on body weight and fat mass, 3-month-old orx mice were treated with the non-aromatizable androgen DHT, E2, or vehicle. The dose of DHT (45 g/d) was chosen to restore the weight of the prostate in orx mice (sham: 10.2 1.0 mg; orx + V: 1.3 0.2 mg; orx + DHT: 8.5 1.0 mg) and the dose of E2 (0.05 g/d) prevented orx-induced trabecular bone loss (trabecular bone mineral density in femur: sham: 281.4 10.7 mg/cm³; orx + V: 197.9 12.5 mg/cm³; orx + E2: 366.0 28.6 mg/cm³). DHT treatment increased body weight, whereas E2 treatment had no significant effect on body weight (Figure 1A). When studying the effect of DHT on different fat depots, the retroperitoneal, brown, and gonadal fat depots weights were increased both in absolute terms and related to body weight (Figure 1 B-D), whereas no significant effect was seen on the inguinal fat depot (data not shown). The effect of DHT on fat mass was associated with an increase in serum leptin levels (Figure 1E). Similar effects of DHT on the fat depots and serum leptin levels were seen in 10-month-old orx mice treated with DHT (data not shown). No major effect of E2 was seen on the weight of the different fat depots or on serum levels of leptin. These findings show that DHT-induced AR activation increases the amount of fat mass in orx male mice.

Figure 1.

DHT increases body weight and fat mass in orx mice. Three-month-old mice were either sham-operated or orx and treated with E2 (0.05 g/d) or DHT (0.45 g/d) for 5 weeks. (A) Body weight, (B) retroperitoneal fat mass/body weight, (C) brown fat mass/body weight, (D) gonadal fat mass/body weight, and (E) serum levels of leptin. Data are presented as mean standard error of the mean. * p < 0.05, ** p < 0.01 vs. orx + vehicle, one-way ANOVA followed by Dunnett's multiple comparison test. n = 11 to 16.

Full figure and legend (112K)

Male ER^{- /-} mice are obese (7, 8) and have elevated serum levels of estrogens and testosterone (19). To determine whether an augmented AR activation might contribute to the obese phenotype in male ER^{- /-} mice, male ER^{- /-} mice were orx and treated with either DHT or E2. Interestingly, orx of the ER^{- /-} mice decreased body mass and the amount of retroperitoneal and brown fat (Figure 2 A-C), indicating that a testicular factor increases the amount of fat in the ER^{- /-} mice. Treatment of 10-month-old orx ER^{- /-} mice with DHT, but not treatment with E2, reversed the loss of fat mass after orx (Figure 2). The effect of DHT on fat mass was associated with an increase in serum leptin levels (Figure 2E). These findings indicate that elevated serum levels of testosterone, resulting in AR activation, contribute to the obese phenotype in male ER^{- /-} mice.

Figure 2.

Effect of DHT on the weight of the fat depots and serum leptin levels in ER^{- /-} mice. Nine-month-old ER^{- /-} mice were either sham-operated or orx and treated with E2 (0.05 g/d) or DHT (0.45 g/d) for 4 weeks. (A) Body weight, (B) retroperitoneal fat mass/body weight, (C) brown fat mass/body weight, (D) gonadal fat mass/body weight, and (E) serum levels of leptin. Data are presented as mean standard error of the mean. ** p < 0.01, * p < 0.05 vs. orx + vehicle, one-way ANOVA followed by Dunnett's multiple comparison test. n = 10 to 14.

Full figure and legend (82K)

To further study whether testosterone treatment affects fat mass through both the AR and the ERs, orx mice were treated with either testosterone or testosterone and an aromatase inhibitor (Figure 3). Testosterone alone did not have any significant effect on the retroperitoneal fat mass, whereas treatment with combined testosterone and aromatase inhibitor increased the retroperitoneal fat mass compared with vehicle-treated orx mice.

Figure 3.

Effect of testosterone (T) and combined testosterone plus aromatase inhibitor (AI) treatment on retroperitoneal fat mass/body weight. Twenty-three-day-old male mice were either sham-operated or orx and treated with T (1.5 g/d) or T + AI (250 g/d) for 5 weeks. Data are presented as mean SE. * p < 0.05 vs. orx + vehicle, one-way ANOVA followed by Dunnett's multiple comparison test. n = 6 to 8.

Full figure and legend (93K)

DHT Treatment Increases RQ

To study the mechanism behind the stimulatory effect of DHT on body weight and fat mass, energy expenditure was measured. Mice were orx and treated with either DHT or vehicle for 1 week. After 1 week of DHT treatment, no significant effect on body weight was apparent. DHT decreased the oxygen consumption (VO₂), whereas it did not have any significant effect on the CO₂ produced (VCO₂; Figure 4 A and B). The decrease in VO₂ but not in VCO₂ resulted in an increased RQ (Figure 4C) and decreased energy expenditure (Figure 4D). Furthermore, when calculating fat and glucose oxidation, it was shown that DHT treatment resulted in a decreased fat oxidation, whereas glucose oxidation was increased (Figure 4 E and F). Changes in body weight may also depend on an altered food consumption and/or locomotor activity. However, DHT did not have any effect on food consumption or urine and feces volume, and no effect of DHT treatment was found on the locomotor activity (data not shown). Thus, the stimulatory effect of DHT on body weight and fat mass is caused by a reduced O₂ consumption associated with a decrease in fat oxidation, whereas no major effect is seen on food consumption or locomotor activity. Given that the fat oxidation was decreased after DHT treatment, we studied whether the gene expression of rate-limiting genes in -oxidation was influenced by DHT treatment in the liver and/or muscle gastrocnemius. However, neither CPT-1 nor medium or long chain acyl-CoA dehydrogenase mRNA levels were significantly influenced by DHT treatment in these tissues (data not shown). Analyses of brown adipose tissue showed that CPT-1 mRNA levels were increased over vehicle (136.5 8.9% , p < 0.01, Student's t test), whereas uncoupling protein 1 levels were unchanged by DHT treatment.

Figure 4.

Effect of DHT on energy expenditure. Three-month-old male mice were orx and treated with DHT (0.45 g/d) for 1 week before measurements. (A) VO₂, (B) VCO₂, (C) RQ, (D) energy expenditure (E) fat oxidation, and (F) glucose oxidation during 2 hours of measurements. Each point represents mean standard error of the mean. n = 8. The steady-state period between 90 and 120 minutes were used for statistical calculations. * p < 0.05, ** p < 0.01 vs. orx + vehicle; two-way ANOVA followed by Student-Newman-Keul's multiple range test.

Full figure and legend (93K)

DHT Treatment Increases High-density Lipoprotein-cholesterol and Triglycerides in orx Mice

DHT treatment increased serum levels of high-density lipoprotein (HDL)-cholesterol and triglycerides (Table 1). The stimulatory effect of DHT on HDL-cholesterol was associated with a trend toward increased total cholesterol (23.7 11.6% over vehicle, p = 0.057). Neither DHT nor E2 affected low-density lipoprotein (LDL)-cholesterol. DHT treatment did not significantly affect insulin (32.5 23.4% over vehicle, not significant) or free fatty acid (11.4 6.9% over vehicle, not significant) levels compared with vehicle-treated orx mice (Table 1).

Table 1 - Effect of DHT and E2 on serum parameters.

Full table

DHT Regulates Hepatic Expression of Genes Involved in Cholesterol and Triglyceride Metabolism

To study the mechanisms behind the effect of DHT on the lipoprotein metabolism, the hepatic expression of several genes involved in regulation of cholesterol and triglyceride metabolism was measured. Cholesterol homeostasis is tightly regulated at several levels, including cholesterol biosynthesis, absorption of dietary cholesterol, cholesterol uptake by the liver, and cholesterol clearance from the liver into bile acids. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase is the rate-limiting enzyme for de novo synthesis of cholesterol in the liver, and its expression is tightly regulated by a feedback mechanism both at the transcriptional and at the translational level (20). The hepatic mRNA expression of 3-hydroxy-3-methylglutarylcoenzyme A reductase was not significantly affected by DHT treatment (Figure 5A). The hepatic uptake of cholesterol from the circulation involves the HDL receptor, scavenger receptor class B member 1, and the low-density lipoprotein receptor (LDLR). Hepatic mRNA expression of scavenger receptor class B member 1 was up-regulated by DHT (45.1 16% ), whereas no significant effect of DHT was seen on hepatic mRNA expression of LDLR (Figure 5 B and C). Because HDL-cholesterol levels were up-regulated by DHT, we studied whether apoA-I, the major apolipoprotein in the HDL fraction, was up-regulated at gene level by DHT. However, neither orx nor treatment of the animals with DHT influenced hepatic apoA-I mRNA expression (Figure 5D). Another gene of importance for HDL turnover is hepatic lipase (21). The various treatments, however, did not influence hepatic lipase mRNA levels (data not shown). The major pathway for excretion of body cholesterol is through the hepatic conversion of cholesterol into bile acids, and it is controlled by the activity of the microsomal enzyme cholesterol 7-hydroxylase. Interestingly, DHT treatment resulted in a pronounced reduction (- 73.0 3.9% ) of cholesterol 7-hydroxylase mRNA levels compared with vehicle-treated orx mice (Figure 5E).

Figure 5.

Effect of DHT on hepatic mRNA levels. Three-month-old mice were either sham-operated or orx and treated with E2 (0.05 g/d) or DHT (0.45 g/d) for 5 weeks. (A) hydroxy-3-methylglutaryl-coenzyme A reductase, (B) scavenger receptor class B member 1, (C) LDLR, (D) apoA-I, (E) cholesterol 7-hydroxylase, (F) stearoyl CoA desaturase-1 (G) microsomal triglyceride transfer protein, and (H) ER. Each mouse was analyzed individually (n = 5), and data are presented as arbitrary units, mean standard error of the mean. * p < 0.05, ** p < 0.01 vs. orx + vehicle, one-way ANOVA followed by Dunnett's multiple comparison test.

Full figure and legend (101K)

Increased serum triglycerides can be a result of either increased VLDL secretion or decreased turnover of VLDL. The finding that DHT increases serum triglycerides indicates increased hepatic triglyceride synthesis, resulting in increased substrate availability for VLDL assembly. Here we show that the hepatic mRNA expression of stearoyl CoA desaturase-1 was increased (16.4 4.1% ; Figure 5F), whereas the mRNA expression of microsomal triglyceride transfer protein was decreased (- 36.1 7.6% ; Figure 5G) by DHT treatment. The mRNA levels of other genes involved in triglyceride biosynthesis and VLDL assembly, glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, acetyl-CoA carboxylase, fatty acid synthase, apolipoprotein AV, or apolipoprotein E were not significantly affected by DHT treatment (data not shown). ApoCII and ApoCIII are of importance for the VLDL turnover. However, the mRNA levels of these genes were not affected by DHT treatment (data not shown).

DHT Treatment Reduces Hepatic ER mRNA Levels

To study whether DHT regulates hepatic sex steroid receptor expression, mRNA levels for AR, ER, and ER were measured. Interestingly, hepatic mRNA expression of ER was clearly reduced by DHT in orx mice (Figure 5H). DHT treatment did not exert any significant effect on hepatic ER or AR mRNA levels (data not shown).

Discussion

It is well known both from clinical and experimental animal studies that sex steroids play an important role in the regulation of fat mass in men. Testosterone and adrenal-derived C19 androgens can be regarded as prohormones, which are converted in the target tissue to DHT or estradiol. The former is the principal ligand for the AR, and the latter is the principal endogenous ligand for the ERs (22). There are, hence, two major pathways, the ER and the AR pathway, for testosterone to influence fat mass. Recent studies have shown that ER activation has the capacity to reduce fat mass in men (7, 8, 23). In this study, we showed, for the first time, that a specific activation of the AR by the non-aromatizable DHT increases fat mass and alters serum lipids in orx mice. A stimulatory effect of a specific AR activation on fat mass is further supported by these findings that testosterone and aromatase inhibitor, but not testosterone alone, increased the amount of fat in orx mice.

To study the mechanism behind the stimulatory effect of DHT on body weight and fat mass, EE, food intake, and physical activity level were measured. DHT-induced obesity was associated with reduced EE but unchanged food intake and spontaneous physical activity. DHT decreased the oxygen consumption, whereas it did not have any significant effect on the CO₂ produced, resulting in increased RQ. Previous studies have shown that high RQ is a predictor of weight gain (24), suggesting that the elevated RQ might be involved in the DHT-induced obesity. The decrease in fat oxidation and the increase in glucose oxidation after DHT treatment are also consistent with early development of obesity (25). Thus, the stimulatory effect of DHT on fat mass is probably caused by reduced EE, associated with a decrease in fat oxidation. The decreased total body fat oxidation was not associated with decreased expression of rate-limiting enzymes in -oxidation in skeletal muscle or the liver, indicating that the decrease in -oxidation was caused by a metabolic effect of DHT on other genes not directly involved in -oxidation, e.g., glucose oxidation. The thermogenic response in brown adipose tissue results in increased expression of uncoupling protein 1 and CPT-1 (26). Increased CPT-1 activity is important for driving brown adipose tissue thermogenesis because it is necessary for the transportation of long-chain fatty acids into mitochondria and subsequent -oxidation. Therefore, the finding of increased CPT-1 expression after DHT treatment could be part of a response to increased -oxidation (26). However, the lack of effect on uncoupling protein 1 expression indicates that the effect of DHT is not a classical thermogenic response. Because brown adipose tissue is probably absent in adult humans, the effects of DHT on brown adipose tissue size and its CPT-1 expression are not relevant to the human situation in terms of an androgen response. One may speculate that the increased CPT-1 levels after DHT treatment were secondary to the elevated leptin levels in the DHT-treated mice.

DHT treatment resulted, in this study, in a pronounced increase in circulating leptin levels. This observation is not surprising, given the established association between obesity and increased production of leptin in most human subjects (27) and most animal models of obesity (28, 29, 30, 31). A role of the AR in the regulation of fat mass is supported by the finding that a CAG repeat polymorphism in the AR is associated with body fat mass and serum levels of leptin in 20- to 50-year-old men (32).

Lack of ER activation, as seen in ER^{- /-} and in aromatase knock-out mice, results in increased fat mass in men (7, 8, 33). Previous studies have interpreted the obese phenotype in these mice to be the result of lack of ER activation alone. However, these mouse models do not only have a lack of ER activation but also have increased serum levels of testosterone, resulting in enhanced AR activation. Interestingly, these findings that orx of the ER^{- /-} mice resulted in a decreased amount of fat indicates that a testicular factor, presumably testosterone, increases the amount of fat in the ER^{- /-} mice. Furthermore, treatment with DHT (AR activation) reversed the loss of fat mass following orx in ER^{- /-} mice. These findings indicate that elevated serum levels of testosterone, resulting in AR activation, might contribute to the obese phenotype in male ER^{- /-} mice and maybe also in male aromatase knock-out mice. A recent report investigating the skeletal phenotype of the male ER-inactivated mice, showing AR-mediated alterations of the trabecular bone, supported the hypothesis that the elevated testosterone levels acting through the AR are of crucial importance for the phenotype in these mice (34). Previous studies have shown that DHT can be converted to 5-androstane-3,17-diol, and it has been shown that 5-androstane-3,17-diol, through activation of ER, exerts antiproliferative effects in the prostate (35). Thus, it could be suggested that DHT after conversion to 5-androstane-3,17-diol regulates fat mass through activation of ER. However, an ER-mediated mechanism is less likely to explain the stimulatory effect of DHT on fat mass seen in this study, because DHT but not estradiol treatment increased fat mass in orx ER-inactivated mice.

Hyperinsulinemic hyperandrogenism with anovulation, the so-called polycystic ovary syndrome, is the most frequent endocrine disorder of young women. One of the stigmata of these hyperandrogenic women is an excess of fat mass, in particular, abdominal fat (36). Interestingly, a combined treatment with AR blockade (flutamide) together with an insulin-sensitizing compound, such as metformin, reduces fat mass in patients with polycystic ovary syndrome (37). These findings, that AR activation increases fat mass, suggests that it is the AR blockade in this combined treatment of polycystic ovary syndrome that it is crucial for the fat-reducing effect. However, the potential use of AR blockade for the reduction of fat mass in obese men is unknown.

DHT treatment resulted in an altered lipid metabolism characterized by increased HDL-cholesterol and serum triglycerides. To our knowledge, this is the first study showing a specific effect of AR activation on serum lipid levels in rodents. The dominant lipoprotein involved in delivery of cholesterol to extrahepatic tissues and the liver is HDL in rodents, whereas low-density lipoprotein serves this role in humans (38, 39). Previous studies showed that aromatizable androgens reduce HDL-cholesterol (40, 41, 42). However, as discussed above regarding the regulation of fat mass, the effect of aromatizable androgens might be exerted through an activation of the ERs rather than through activation of the AR. A role of the AR in the regulation of HDL-cholesterol is supported by the finding of a positive relation of the number of CAG repeats in the AR with HDL levels in men (43).

To study the mechanism of action for the DHT-induced increase in HDL-cholesterol, mRNA levels of key genes implicated in the cholesterol homeostasis were measured. Hence, the mRNA levels of genes regulating 1) cholesterol synthesis, 2) clearance of plasma lipoproteins through receptors, 3) degradation of cholesterol into bile acids, and 4) secretion of lipoprotein into the plasma compartment were measured. SR-BI is a scavenger receptor that in the liver is involved in selective uptake of cholesterol esters from HDL particles, one of the key steps in reverse cholesterol transport (44, 45). In accordance with previous in vitro studies of hepatocytes treated with testosterone, we showed a modest increase in scavenger receptor class B member 1 mRNA expression in the liver after DHT treatment (46). The elevated scavenger receptor class B member 1 mRNA levels indicate increased hepatic uptake of cholesterol esters as a part of the reverse cholesterol transport in DHT-treated mice that would result in decreased serum levels of HDL-cholesterol. Therefore, other mechanisms must explain the increased serum levels of HDL-cholesterol. Another possibility is increased production of apoA-I, which is the major apolipoprotein of HDL. However, we found no regulation of apoA-I mRNA expression by DHT treatment. A third possibility is reduced activity of hepatic lipase because hepatic lipase is important for hydrolysis of lipids in the HDL fraction (21). However, hepatic lipase was not influenced by orx or DHT treatment. A fourth possibility, explaining increased serum HDL, is increased production and turnover of VLDL. There are no studies on the effects of DHT on VLDL metabolism. However, studies in rats have shown that testosterone decreases (47), whereas estradiol increases VLDL production (48). In this study, hepatic mRNA expression of stearoyl CoA desaturase-1 was increased, whereas microsomal triglyceride transfer protein mRNA expression was decreased by DHT treatment. Increased microsomal triglyceride transfer protein expression has been shown to increase apoB and VLDL secretion (49), indicating that the decreased microsomal triglyceride transfer protein expression as a result of DHT treatment would result in decreased VLDL secretion. Stearoyl CoA desaturase-1 is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids and of importance for hepatic triglyceride biosynthesis (50). Mice lacking stearoyl CoA desaturase-1 are lean as a result of increased EE (51). Thus, stearoyl CoA desaturase-1 has been proposed as a molecular target for the treatment of obesity, and it is known that leptin regulates the stearoyl CoA desaturase-1 expression (52). One may speculate that the increased leptin levels in the DHT-treated mice might explain the increase in hepatic stearoyl CoA desaturase-1 expression. However, a major effect of the rather small increase in stearoyl CoA desaturase-1 gene expression seen after DHT treatment on hepatic triglyceride biosynthesis or triglyceride secretion is unlikely.

Importantly, DHT treatment resulted in a pronounced decrease in hepatic cholesterol 7-hydroxylase mRNA expression. Cholesterol 7-hydroxylase is the key enzyme in the regulation of cholesterol conversion into bile acids, which is the major pathway whereby cholesterol is removed from the body (53). The observed decrease in cholesterol 7-hydroxylase expression would result in decreased cholesterol removal from the liver and, in contrast to the increased scavenger receptor class B member 1 expression, would result in decreased reverse cholesterol transport. Therefore, it would be anticipated that cholesterol accumulates in the liver cells and decreases the cleavage and activation of sterol regulatory element-binding protein 2 that activates transcription of the low-density lipoprotein receptor and hydroxy-3-methylglutaryl-coenzyme A reductase. In line with this assumption, a trend toward a decreased expression of these genes was observed in this study.

For some of the parameters studied, including HDL-cholesterol, a significant effect of DHT treatment was seen, whereas a tendency toward an opposite effect was seen after E2 treatment. Interestingly, the hepatic mRNA expression of ER was clearly reduced by DHT in orx mice. Hence, it cannot be excluded that some of the hepatic effect of DHT, seen in this study, is mediated through a reduced ER activity.

In conclusion, we showed that AR activation resulted in obesity and disturbed lipid metabolism in orx mice. One may speculate that AR antagonists might be useful in the treatment of obesity in men.

Notes

¹ Nonstandard abbreviations: AR, androgen receptor; ER, estrogen receptor; orx, orchidectomized; DHT, 5-dihydrotestosterone; E2, 17-estradiol; RQ, respiratory quotient; EE, energy expenditure; V, vehicle; CPT-1, carnitine palmitoyltransferase-1; HDL, high-density lipoprotein; LDLR, low-density lipoprotein receptor; SEM, standard error of the mean.

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Acknowledgments

We thank Anette Hansevi and Maud Petersson for excellent technical assistance. This study was supported by the Swedish Medical Research Council, the Swedish Foundation for Strategic Research, European Commission Grant QLK4-CT-2002-02,528, the Lundberg Foundation, the Novo Nordisk Foundation, the Torsten and Ragnar Söderberg's Foundation, and Petrus and Augusta Hedlund's Foundation.