**2. Materials and Methods**

### **2.1. Animals**

Male Wistar rats and C57/black mice at 8 wk of age were fed with or without (control) 0.4% testosterone or 0.4% DHEA containing food in CE2 powder (carbohydrate 51.4%, protein 24.9%, fat 4.6%, fiber 3.7%) for 4 wk. Individual food consumption was deter‐ mined by subtracting the food remaining from that supplied every 2-3 days, with the averages of these values in one week expressed as the weekly food consumption. The animals were housed in a specific pathogen-free facility with a 12-h light/12-h dark cycle. After sacrifice white adipose tissue (epididymal fat), skeletal muscle (gastrocnemius muscle) brown adipose tissue (BAT) and liver were collected. All procedures for animal care were carried out in accordance with protocols approved by the University of Gifu's Institution‐ al Animal Care Committee.

O2 consumption (VO2), CO2 production (VCO2) and locomotor activity in mice were measured individually by indirect calorimetry using an Oxymax apparatus (Columbus Instruments, Columbus, OH) as described previously [42]. Respiratory exchange rate (RER) was calculated as VCO2/VO2. Heat generation was calculated as caloric value (3.815+1.232 × RER) × VO2.

#### **2.2. Cell culture**

3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Upon confluence, 3T3-L1 preadipocytes were differentiated with differentiation medium containing insulin, dexamethasone and IBMX for 3 days followed by incubation with DMEM again. At 5 days after the differentiation, cells were stimulated with 50 nM DHEA or 50 nM testosterone for 48 hr. The content of triglyceride was visualized with Oil Red O (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer's instruction.

F442A preadipocytes were cultured in DMEA with supplement as described above. When confluence was reached (0d), 50 nM DHEA or testosterone was added to the medium to evaluate the effects of these hormones on spontaneous differentiation of F442A cells into mature adipocytes without differentiation medium.

C2C12 myoblasts were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ mL penicillin, and 0.1 mg/mL streptomycin. When cells reached 90% confluence, the medium was exchanged for DMEM containing 4% horse serum (differentiation medium). After incubation with the differentiation medium for 7 days, cells were morphologically determined to complete the differentiation into C2C12 myotubes, and then these cells were treated with various concentrations of testosterone for 48 hr

#### **2.3. Real time PCR**

Real time PCR was performed to measure mRNA expression levels of PPARγ, fatty acid binding protein 4 (FABP4), lipoprotein lipase (LPL), adiponectin, SREBP-1, fatty acid synthase (FAS) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) in 3T3-L1 adipocytes, and PGC1α, cytochrome C and G3PDH in C2C12 myotubes, as described previously [43-45]. All data were normalized to the expression level of G3PDH.

#### **2.4. Triglyceride content in liver and skeletal muscle**

Liver and gastrocunemius muscle were homogenized in KRP buffer, and the triglyceride in the homogenate was extracted with chloroform-methanol, and assayed using a LabAs‐ say Triglyceride kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan) as described previously [43].

#### **2.5. Western blot**

**2. Materials and Methods**

al Animal Care Committee.

**2.2. Cell culture**

**2.3. Real time PCR**

Male Wistar rats and C57/black mice at 8 wk of age were fed with or without (control) 0.4% testosterone or 0.4% DHEA containing food in CE2 powder (carbohydrate 51.4%, protein 24.9%, fat 4.6%, fiber 3.7%) for 4 wk. Individual food consumption was deter‐ mined by subtracting the food remaining from that supplied every 2-3 days, with the averages of these values in one week expressed as the weekly food consumption. The animals were housed in a specific pathogen-free facility with a 12-h light/12-h dark cycle. After sacrifice white adipose tissue (epididymal fat), skeletal muscle (gastrocnemius muscle) brown adipose tissue (BAT) and liver were collected. All procedures for animal care were carried out in accordance with protocols approved by the University of Gifu's Institution‐

O2 consumption (VO2), CO2 production (VCO2) and locomotor activity in mice were measured individually by indirect calorimetry using an Oxymax apparatus (Columbus Instruments, Columbus, OH) as described previously [42]. Respiratory exchange rate (RER) was calculated as VCO2/VO2. Heat generation was calculated as caloric value (3.815+1.232 × RER) × VO2.

3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Upon confluence, 3T3-L1 preadipocytes were differentiated with differentiation medium containing insulin, dexamethasone and IBMX for 3 days followed by incubation with DMEM again. At 5 days after the differentiation, cells were stimulated with 50 nM DHEA or 50 nM testosterone for 48 hr. The content of triglyceride was visualized with Oil Red O (Santa Cruz

F442A preadipocytes were cultured in DMEA with supplement as described above. When confluence was reached (0d), 50 nM DHEA or testosterone was added to the medium to evaluate the effects of these hormones on spontaneous differentiation of F442A cells into

C2C12 myoblasts were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ mL penicillin, and 0.1 mg/mL streptomycin. When cells reached 90% confluence, the medium was exchanged for DMEM containing 4% horse serum (differentiation medium). After incubation with the differentiation medium for 7 days, cells were morphologically determined to complete the differentiation into C2C12 myotubes, and then these cells were treated with

Real time PCR was performed to measure mRNA expression levels of PPARγ, fatty acid binding protein 4 (FABP4), lipoprotein lipase (LPL), adiponectin, SREBP-1, fatty acid synthase

Biotechnology, Santa Cruz, CA) according to the manufacturer's instruction.

mature adipocytes without differentiation medium.

various concentrations of testosterone for 48 hr

**2.1. Animals**

298 Treatment of Type 2 Diabetes

The cell lysate was mixed with Laemmli sample buffer and boiled for 3 min. Equal amounts of cell lysate were subjected to SDS-PAGE, and transferred onto nitrocellulose paper. The paper was blocked with 1% BSA, and incubated with anti-PPARγ antibody, anti-adiponectin antibody or anti-actin antibody (Santa Cruz). Protein bands were visualized with an ECL system.

#### **2.6. Statistics**

All experimental results were calculated as means ± SE. Statistical comparisons were per‐ formed by Student's t-test or ANOVA. Significance was defined as *P* < 0.05.
