**3. Results**

#### **3.1. Body weight and plasma glucose level**

Treatment with testosterone or DHEA containing food reduced weight gain in both rats (Fig. 1A) and mice (Fig. 1B). Administration of testosterone and DHEA reduced body weight equivalently. The dose response study showed that food containing both testosterone and DHEA at 0.4% significantly suppressed body weight gain (Fig.1C). Our previous study [43] indicated that treatment with 0.4% testosterone for 4 wk resulted in an increase of serum testosterone and DHEA-S levels up to 674% and 1040%, respectively (note that serum DHEA-S level is very low in rodents due to the lack of 17α hydroxylase in adrenal glands), whereas treatment with DHEA increased testosterone and DHEA-S levels up to 310% and 6420%, respectively. The fact that these androgens are convertible to each other, partially explains the similar results obtained with administration of these hormones. Administration of testosterone and DHEA did not influence fasting plasma glucose level in rats (Fig. 1D), while testosterone suppressed it a little but significantly in mice (Fig. 1E). Food consumption was not influenced by the administration of either hormone in rats (Fig. 1F).

Fig. 1 **Figure 1.** Effects of administration of DHEA and testosterone on body weight. Effects of treatment with 0.4% DHEA or testosterone containing food for 4wk on body weight in Wistar rats (n=6) (A) and C56/black mice (n=4) (B) at 8 wk of age are shown. \*: p<0.05 vs control. Effects of 0.1%-0.4% DHEA or testosterone containing food for 4wk on body weight in C57/black mice (n=4) (C) are shown. \*: p<0.05 vs control. Effects of treatment with 0.4% DHEA or testoster‐ one containing food for 4wk on fasting plasma glucose level in Wistar rats (n=6) (D) and C56/black mice (n=4) (E) are shown. \*: p<0.05 vs control. Effects of DHEA or testosterone administration on food consumption in mice (n=4) are shown. Black solid line: Control, Red broken line: DHEA, Blue broken line: testosterone. \*: p<0.05 vs control.

#### **3.2. Effect of DHEA and testosterone on adipocytes**

Administration of DHEA or testosterone suppressed fat weight, including that of subcutane‐ ous, epididymal and mesenteric fat (Fig. 2A). In addition, both DHEA and testosterone decreased adipocyte size equivalently (Fig. 2B). We found that treatment with DHEA reduced the expression of PPARγ in adipocytes in both *in vivo* and *in vitro* [42]. Treatment with DHEA and testosterone similarly reduced the expression level of PPARγ in adipose tissue isolated from Wistar rats and 3T3-L1 adipocytes (Fig. 2C, D). Genes regulated by PPARγ, such as FABP 4, LPL and adiponectin were equally down-regulated by DHEA and testosterone in 3T3-L1 adipocytes. Neither hormone influenced the expression levels of genes, which are not directly regulated by PPARγ, such as SREBP-1 and FAS (data not shown). Administration of DHEA or testosterone decreased triglyceride content in liver and skeletal muscle to the same degree in rats (Fig. 2 E, F).

**Figure 2.** Effects of administration of DHEA and testosterone on adipocytes. Effects of treatment with DHEA or testos‐ terone for 4wk on fat weight (black: subcutaneous, green: epididymal, blue: mesenteric fat, n=6) (A), and histological findings (B) are shown. \*: p<0.05 vs each control. Effects of treatment with DHEA or testosterone on the protein level of PPARγ and adiponectin in adipose tissue in Wistar rats were evaluated. A typical result was shown (C). Effects of treatment with DHEA or testosterone on the expression of adipocyte specific genes in 3T3-L1 adipocytes are shown (D). Fully differentiated 3T3-L1 adipocytes were incubated with 50 nM DHEA or testosterone for 48 hr. Expression lev‐ els of PPARγ (black), FABP4 (blue), LPL (red), adiponectin (green) were shown (n=6). \*: p<0.05 vs each control. Effects of treatment with DHEA or testosterone on triglyceride content in liver (n=5) (E) and skeletal muscle (n=5) (F) in Wistar rats are presented. \*:p<0.05 vs control.

Next, we examined the effects of these hormones on adipocyte differentiation. We observed the differentiation of F442A cells, since they spontaneously differentiate into mature adipo‐ cytes when they reach confluence. DHEA and testosterone suppressed the accumulation of triglyceride (Fig. 3A) and the appearance of PPARγ and FABP4 mRNA during the differen‐ tiation process. These data indicated that DHEA and testosterone similarly suppress adipocyte differentiation.

#### **3.3. Effect of DHEA and testosterone on mitochondrial biogenesis**

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Fig. 1 **Figure 1.** Effects of administration of DHEA and testosterone on body weight. Effects of treatment with 0.4% DHEA or testosterone containing food for 4wk on body weight in Wistar rats (n=6) (A) and C56/black mice (n=4) (B) at 8 wk of age are shown. \*: p<0.05 vs control. Effects of 0.1%-0.4% DHEA or testosterone containing food for 4wk on body weight in C57/black mice (n=4) (C) are shown. \*: p<0.05 vs control. Effects of treatment with 0.4% DHEA or testoster‐ one containing food for 4wk on fasting plasma glucose level in Wistar rats (n=6) (D) and C56/black mice (n=4) (E) are shown. \*: p<0.05 vs control. Effects of DHEA or testosterone administration on food consumption in mice (n=4) are

Administration of DHEA or testosterone suppressed fat weight, including that of subcutane‐ ous, epididymal and mesenteric fat (Fig. 2A). In addition, both DHEA and testosterone decreased adipocyte size equivalently (Fig. 2B). We found that treatment with DHEA reduced the expression of PPARγ in adipocytes in both *in vivo* and *in vitro* [42]. Treatment with DHEA and testosterone similarly reduced the expression level of PPARγ in adipose tissue isolated from Wistar rats and 3T3-L1 adipocytes (Fig. 2C, D). Genes regulated by PPARγ, such as FABP 4, LPL and adiponectin were equally down-regulated by DHEA and testosterone in 3T3-L1 adipocytes. Neither hormone influenced the expression levels of genes, which are not directly regulated by PPARγ, such as SREBP-1 and FAS (data not shown). Administration of DHEA or testosterone decreased triglyceride content in liver and skeletal muscle to the same degree

shown. Black solid line: Control, Red broken line: DHEA, Blue broken line: testosterone. \*: p<0.05 vs control.

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As noted above, since the administration of neither DHEA nor testosterone influenced food consumption, we speculated that these hormones elevate energy expenditure. Hence we examined the effects of testosterone administration on energy production. Mice were treated with or without testosterone for 4 wk, and then, oxygen consumption and locomotor activity were measured by indirect calorimetry. O2 consumption and CO2 production were increased A

**Figure 3.** Effect of treatment with DHEA and testosterone on the differentiation of F442A adipocytes. F442A preadipo‐ Fig. 3 cytes were cultured in DMED. When cells reached confluence as judged by the morphological findings (0d), 50nM DHEA or testosterone was added to the medium, followed by subsequent incubation for the indicated period. Trigly‐ ceride accumulation was assessed with oil-Red staining at 7d (A). Expression levels of PPARγ and FABP4 were meas‐ ured with real time PCR on the indicated day (n=4) (B). \*:p<0.05 vs each control.

significantly in testosterone-treated mice, regardless of whether the values were normalized by body weight or not (Fig. 4B-E). In addition, heat production, the values of which were normalized by body weight, was elevated in testosterone-treated mice (Fig. 4G). No difference was detected in respiratory exchange rate between control and testosterone-treated mice (Fig. 4H). To our surprise, administration of testosterone suppressed locomotor activity (Fig. 4I).

These results indicate that administration of testosterone increases the basal metabolic rate. Therefore, we evaluated the effects of administration of these androgens on mitochondri‐ al biogenesis and its upstream regulator, PGC1α. Expression of mitochondrial protein, Cox4, and PGC1α was elevated in skeletal muscle, but not brown BAT or liver, isolated from testosterone-treated rats (Fig. 5A). The increase of Cox4 in skeletal muscle induced by DHEA administration was less than that induced by testosterone (Fig. 5B). The testosteroneinduced increases in mRNA levels of PGC1α and cytochrome C were greater than the DHEA-induced ones in C2C12 myotubes (Fig. 5C). These results show that increased mitochondrial biogenesis by these hormones leads to up-regulation of energy expendi‐ ture, which may result in reduced adiposity.

Anti-Obesity Effects of Androgens, Dehydroepiandrosterone (DHEA) and Testosterone http://dx.doi.org/10.5772/59604 303

significantly in testosterone-treated mice, regardless of whether the values were normalized by body weight or not (Fig. 4B-E). In addition, heat production, the values of which were normalized by body weight, was elevated in testosterone-treated mice (Fig. 4G). No difference was detected in respiratory exchange rate between control and testosterone-treated mice (Fig. 4H). To our surprise, administration of testosterone suppressed locomotor activity (Fig. 4I).

**Figure 3.** Effect of treatment with DHEA and testosterone on the differentiation of F442A adipocytes. F442A preadipo‐ Fig. 3 cytes were cultured in DMED. When cells reached confluence as judged by the morphological findings (0d), 50nM DHEA or testosterone was added to the medium, followed by subsequent incubation for the indicated period. Trigly‐ ceride accumulation was assessed with oil-Red staining at 7d (A). Expression levels of PPARγ and FABP4 were meas‐

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These results indicate that administration of testosterone increases the basal metabolic rate. Therefore, we evaluated the effects of administration of these androgens on mitochondri‐ al biogenesis and its upstream regulator, PGC1α. Expression of mitochondrial protein, Cox4, and PGC1α was elevated in skeletal muscle, but not brown BAT or liver, isolated from testosterone-treated rats (Fig. 5A). The increase of Cox4 in skeletal muscle induced by DHEA administration was less than that induced by testosterone (Fig. 5B). The testosteroneinduced increases in mRNA levels of PGC1α and cytochrome C were greater than the DHEA-induced ones in C2C12 myotubes (Fig. 5C). These results show that increased mitochondrial biogenesis by these hormones leads to up-regulation of energy expendi‐

ture, which may result in reduced adiposity.

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302 Treatment of Type 2 Diabetes

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**Figure 4.** Effects of treatment with testosterone on oxygen consumption, heat production and locomotor activity. C56/ black mice at 8 wk of age were treated with testosterone for 4 wk, and individual oxygen consumption and locomotor activity were determined by indirect calorimetry (A). Cumulative O2 consumption for 24 hr (B) and normalized values by body weight (C), CO2 production (D) and normalized values by body weight (E), heat production for 24 hr (F) and normalized values by body weight (G) are shown. Values of RER (H) and locomotor activity (I) for 24 hr are also shown. \*: p<0.05 vs control, \*\*: p<0.01 vs control

**Figure 5.** Effect of treatment with DHEA and testosterone on mitochondrial biogenesis. Wistar rats were treated with DHEA or testosterone for 4 wk. Effects of treatment with testosterone on the expression of PGC1α and Cox4 in skeletal muscle, BAT and liver are shown (A). Typical results of western blot are shown in the left panel, and quantified results are shown in the right (n=4). White: Control, Black: Testosterone-treated. \*:p<0.05 vs control. Representative image of immunohistochemistry of skeletal muscle isolated from control, DHEA-treated and testosterone-treated rats are shown (B). Effects of incubation with 10 nM DHEA or testosterone for 48 hr on the expression of PGC1α and cytochrome C mRNA in C2C12 myotubes (n-4) are shown (C). \*: p<0.05 vs control, #: p<0.05 vs DHEA.

#### **4. Discussion**

Coleman *et al*., demonstrated that administration of DHEA reduces blood glucose level in db/ db mice [46]. We found that administration of DHEA improved blood glucose in OLETF rats, a model of obese diabetes, but not GK rats, a model of lean diabetes [47]. Accordingly, we presumed that DHEA-induced weight reduction might contribute to improving blood glucose levels. Although administration of DHEA consistently suppresses body weight and fat weight, significant improvement of blood glucose is detected only in extremely obese animals [44]. We noted that DHEA and testosterone reduce the expression of PPARγ in adipocytes [43, 44]. Heterozygous PPARγ deficient mice are protected from insulin resistance under a high-fat diet [48], and reduced receptor activity of PPARγ by Pro12Ala substitution leads to lower body mass index in man [49], suggesting that modest suppression of PPARγ activity may help to prevent obesity and resultant insulin resistance. However, the production and secretion of adiponectin are positively regulated by PPARγ in adipocytes [50], and inhibition of PPARγ may result in insulin resistance due to low plasma adiponectin level. In this study, we showed that DHEA and testosterone decrease PPARγ, as well as adiponectin (Fig. 2C, D). This result is consistent with the fact that despite their obese phenotype, glucose homeostasis remained intact because of a high plasma adiponectin level in androgen receptor null mice (ARKO) [51]. These data explain the results of the numerous clinical studies described above in which administration of DHEA or testosterone consistently reduced adiposity, despite which numerous studies have failed to find proof of any beneficial effect on glucose metabolism.

This study confirmed that administration of DHEA and testosterone reduced body weight and fat weight equally, as described in our previous study [44]. If this conclusion is applied to men for weight reduction, supplementation of DHEA would be more desirable than that of testosterone given the smaller possibility of adverse effects. Our study also reveals that DHEA and testosterone attenuate proliferation of 3T3-L1 preadipocytes in a similar concentration dependent manner [44]. In addition, we showed that these hormones decrease the expression levels of PPARγ, LPL and FABP4, but not SREBP-1, at common concentrations and in a time dependent manner [44]. The possibility that fat content increased in other organs in compen‐ sation for the decrease in fat mass, was ruled out by the fact that fat content in liver and skeletal muscle decreased similarly both in DHEA and testosterone-treated rats [44], which was confirmed in this experiment. The findings that neither DHEA nor testosterone increased glycerol release in 3T3-L1 adipocytes and administration of these hormones decreased serum free fatty acid concentration in rats, rule out the possibility that these hormones reduce adiposity by increased lipolysis [41]. In this study, we revealed that both DHEA and testos‐ terone suppress differentiation of adipocytes using F442A. Both DHEA and testosterone equivalently inhibited spontaneous differentiation of cells. Recently, concurrent results have been published with regard to 3T3-L1 preadipocytes, C3H 10T1/2 pluripotent cells and human preadipocytes [52-55]. Singh *et al,* reported that formation of androgen receptor/β-catenin and T-cell factor 4 complex and activation of Wnt signaling are involved in androgen-induced inhibition of adipogenesis [54].

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**Figure 5.** Effect of treatment with DHEA and testosterone on mitochondrial biogenesis. Wistar rats were treated with DHEA or testosterone for 4 wk. Effects of treatment with testosterone on the expression of PGC1α and Cox4 in skeletal muscle, BAT and liver are shown (A). Typical results of western blot are shown in the left panel, and quantified results are shown in the right (n=4). White: Control, Black: Testosterone-treated. \*:p<0.05 vs control. Representative image of immunohistochemistry of skeletal muscle isolated from control, DHEA-treated and testosterone-treated rats are shown (B). Effects of incubation with 10 nM DHEA or testosterone for 48 hr on the expression of PGC1α and cytochrome C

Coleman *et al*., demonstrated that administration of DHEA reduces blood glucose level in db/ db mice [46]. We found that administration of DHEA improved blood glucose in OLETF rats, a model of obese diabetes, but not GK rats, a model of lean diabetes [47]. Accordingly, we presumed that DHEA-induced weight reduction might contribute to improving blood glucose levels. Although administration of DHEA consistently suppresses body weight and fat weight, significant improvement of blood glucose is detected only in extremely obese animals [44]. We noted that DHEA and testosterone reduce the expression of PPARγ in adipocytes [43, 44]. Heterozygous PPARγ deficient mice are protected from insulin resistance under a high-fat diet [48], and reduced receptor activity of PPARγ by Pro12Ala substitution leads to lower body mass index in man [49], suggesting that modest suppression of PPARγ activity may help to

\*

**PGC1alpha (Control: 100%)**

To clarify the mechanisms underlying androgen-induced weight reduction, we analyzed the effect of testosterone administration on energy expenditure. Administration of both DHEA and testosterone increased the rectal temperature in rats [44]. Although an abnormally high body temperature was not detected, elevated O2 consumption and CO2 production was observed in testosterone-treated mice (Fig. 4A-D). Although heat production was increased in testosterone-treated mice, it was not significant when these values were not normalized by body weight (Fig. 4E). We have no data on lean body mass or water. If lean body mass is not influenced by testosterone, testosterone-induced reduction of adiposity could not result from an increase in energy expenditure. On the other hand, our results indicate that basal metabolic rate increases in testosterone-treated mice since heat production in these mice did not decreas despite suppressed locomotor activity. The result of suppressed locomotor activity in testos‐ terone-treated mice was unexpected, since lower locomotor activity was also reported in ARKO [51]. We are not yet able to explain this discrepancy, probably because change in locomotor activity may not occur in parallel with an androgen signal.

Next, we speculated that testosterone might increase mitochondrial activity to explain the increased basal metabolic rate. As shown in Fig. 5A, increased Cox4, a mitochondrial protein, as well as PGC1α, an up-stream regulator of mitochondrial biogenesis, was recognized in skeletal muscle isolated from testosterone-treated rats. Similar results were noted in mice [45]. In addition, treatment with testosterone up-regulates the expression levels of genes contribu‐ ting to mitochondrial biogenesis, such as nuclear respiratory factor-1 (NRF-1), NRF-2 and mitochondrial transcriptional factor A (Tfam), as well as mitochondrial DNA (mitDNA) in skeletal muscle [44]. Although DHEA and testosterone exhibit similar effects on adipocytes, administration of DHEA resulted in less increase in Cox4 than that of testosterone in skeletal muscle. This result was confirmed by the experiment showing that the testosterone-induced increase in mRNA of PGC1α and cytochrome C was greater than the DHEA-induced ones (Fig. 5C) in C2C12 myotubes. These results are consistent with data published by Sato *et al.* [41]. These differences in the response to DHEA and testosterone between adipocytes and myocytes may be attributable to differences in the efficacy of subcellular steroid converting enzymes. Although we did not assess the effect of androgens on total skeletal muscle volume, androgens have been reported to enhance the differentiation into skeletal muscle [53]. Therefore, the conclusion derived from our experiment should be further explored by increasing the whole skeletal muscle mass. In addition, we found that expression of PGC1α and mitochondrial genes was reduced in skeletal muscle isolated from ARKO [45].

The results of our studies were summarized in Fig. 6. DHEA and testosterone equally sup‐ pressed proliferation of preadipocytes, differentiation of adipocytes and expression of PPARγ and its down-stream genes including adiponectin in adipocytes. Both DHEA and testosterone up-regulated PGC1α and mitochondrial biogenesis, more actively in the latter than the former in skeletal muscle. Which organ plays the main role in the androgens-induced reduction of adiposity remains an interesting problem. Our results suggest that reduced adiposity in testosterone-treated animals may be derived from decreased expression of PPARγ and suppressed differentiation into adipocytes. Moderate suppression of PPARγ activity by its antagonist HX531 resulted in decreased fat mass and increased oxygen con‐ sumption [56], and therefore androgen-induced reduction of PPARγ expression may be able to influence systemic energy metabolism.

Whole body silencing of AR results in late-onset obesity [51, 56]. Recent technology has facilitated the generation of organ specific deletion of a gene. Adipocyte specific AR deficient mice showed identical body weight and adiposity with wild type at 20 wk of age in one study, although the authors did not show the data of older mice [57]. Since late obesity after 20 wk of age is the distinguishing feature in ARKO, this point is important. Conversely, mice lacking AR in the central nervous system develop late onset obesity and insulin resistance [59]. Although several investigations have reported that myocyte specific AR knockdown did not influence body weight and adiposity [60, 61], myocyte specific AR overexpression resulted in an increased metabolic rate and fat body mass [62]. These results suggest that skeletal muscle and brain might be responsible organs for androgen-induced reduction of adiposity. However,

**Figure 6.** Effect of DHEA and testosterone on brain, adipocytes and skeletal muscle

the role of AR in adipocytes in systemic insulin sensitivity cannot be ruled out at present. Further experiments will be required to help clarify these issues.
