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Am J Physiol Endocrinol Metab 294: E961-E968, 2008. First published March 18, 2008; doi:10.1152/ajpendo.00678.2007
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Testosterone and DHEA activate the glucose metabolism-related signaling pathway in skeletal muscle

Koji Sato,1 Motoyuki Iemitsu,2 Katsuji Aizawa,1 and Ryuichi Ajisaka1

1Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki; and 2Department of Physical Education, International Pacific University, Okayama City, Okayama, Japan

Submitted 23 October 2007 ; accepted in final form 10 March 2008


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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 REFERENCES
 
Circulating dehydroepiandrosterone (DHEA) is converted to testosterone or estrogen in the target tissues. Recently, we demonstrated that skeletal muscles are capable of locally synthesizing circulating DHEA to testosterone and estrogen. Furthermore, testosterone is converted to 5{alpha}-dihydrotestosterone (DHT) by 5{alpha}-reductase and exerts biophysiological actions through binding to androgen receptors. However, it remains unclear whether skeletal muscle can synthesize DHT from testosterone and/or DHEA and whether these hormones affect glucose metabolism-related signaling pathway in skeletal muscles. We hypothesized that locally synthesized DHT from testosterone and/or DHEA activates glucose transporter-4 (GLUT-4)-regulating pathway in skeletal muscles. The aim of the present study was to clarify whether DHT is synthesized from testosterone and/or DHEA in cultured skeletal muscle cells and whether these hormones affect the GLUT-4-related signaling pathway in skeletal muscles. In the present study, the expression of 5{alpha}-reductase mRNA was detected in rat cultured skeletal muscle cells, and the addition of testosterone or DHEA increased intramuscular DHT concentrations. Addition of testosterone or DHEA increased GLUT-4 protein expression and its translocation. Furthermore, Akt and protein kinase C-{zeta}/{lambda} (PKC-{zeta}/{lambda}) phosphorylations, which are critical in GLUT-4-regulated signaling pathways, were enhanced by testosterone or DHEA addition. Testosterone- and DHEA-induced increases in both GLUT-4 expression and Akt and PKC-{zeta}/{lambda} phosphorylations were blocked by a DHT inhibitor. Finally, the activities of phosphofructokinase and hexokinase, main glycolytic enzymes, were enhanced by testosterone or DHEA addition. These findings suggest that skeletal muscle is capable of synthesizing DHT from testosterone, and that DHT activates the glucose metabolism-related signaling pathway in skeletal muscle cells.

5{alpha}-dihydrotestosterone; dehydroepiandrosterone; glucose transporter-4; Akt; protein kinase C-{zeta}/{lambda}

SEX STEROID HORMONES ARE MAINLY produced and secreted by the ovary, testis, and adrenal cortex and affect diverse physiological processes of target organs or tissues, such as reproductive organs, bones, liver, heart, vasculature, brain, and skeletal muscles (22). Synthesis of testosterone is regulated by P-450 side-chain cleavage, 17{alpha}-hydroxylase cytochrome P-450, 17β-hydroxysteroid dehydrogenase (HSD), and 3β-HSD enzymes. Dehydroepiandrosterone (DHEA) is a presubstance of sex steroid hormones, and DHEA is converted to testosterone by 17β-HSD and 3β-HSD enzymes (23). Recently, our laboratory found that 3β-HSD, 17β-HSD, and aromatase cytochrome P-450 existed in cultured skeletal muscle and steroid hormones, including testosterone, and were locally synthesized from DHEA (1). In a skeletal muscle specimen, testosterone administration accelerated protein synthesis (1), resulting in muscle growth and/or increased strength (30). To exert biophysiological actions through binding to androgen receptors, testosterone is converted to 5{alpha}-dihydrotestosterone (DHT) by 5{alpha}-reductase type 1 and type 2 enzymes (8). However, the existence of 5{alpha}-reductase enzymes in skeletal muscle has not been clarified. Therefore, it remains unclear whether DHT can be converted from testosterone or DHEA in skeletal muscle.

In the regulation of glucose transporter-4 (GLUT-4), it is pivotal that insulin binding receptor substrate upregulates the activation of protein kinase B (Akt) and/or protein kinase C-{zeta}/{lambda} (PKC-{zeta}/{lambda}) via phosphatidylinositol 3-kinase (PI 3-kinase) in skeletal muscles (9, 17). Recently, in osteoblast cells, Kang et al. (18) reported that testosterone induced the activation of PI 3-kinase, resulting in accelerated Akt phosphorylation, and the inhibition of PI 3-kinase abolished DHT-induced Akt activation. Therefore, testosterone or DHT may activate these glucose metabolism-related signaling pathways in skeletal muscles. However, the effects of locally synthesized sex steroid hormones on glucose metabolism in skeletal muscles remain unclear.

Subsequently, we hypothesized that DHT was locally synthesized from testosterone or DHEA and activated the glucose metabolism-related signaling pathway in skeletal muscle. To test these hypotheses, we first investigated whether 5{alpha}-reductase enzymes exist in cultured skeletal muscle, and whether skeletal muscle could synthesize DHT from testosterone and/or DHEA. Second, we examined whether testosterone and/or DHEA upregulates the signaling pathway of Akt and PKC-{zeta}/{lambda} phosphorylations with GLUT-4 protein expression and translocation. Furthermore, we examined the activities of the two main glycolytic enzymes [phosphofructokinase (PFK) and hexokinase (HK)] in the skeletal muscle. Finally, we confirmed whether the effects of DHT, locally synthesized from testosterone and/or DHEA, on the GLUT-4-related signaling pathway were inhibited by a 5{alpha}-reductase blocker, which inhibits the synthesis of DHT. To test our hypotheses, the present study used a primary culture in the skeletal muscle cells of neonatal rats.


   METHODS
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Incubated skeletal muscle cells. Myoblasts of skeletal muscle were isolated from 2- to 3-day-old Sprague-Dawley rats, according to the method described in previous reports (1, 28). After confluent proliferation for 1 day, myoblast cells were incubated on fibronectin-coated dishes in Dulbecco's modified Eagle's medium/Ham's F-12 medium, supplemented with 2 ml of Dulbecco's modified Eagle's medium + 2% horse serum (Cell Garage, Hokkaido, Japan) for 7 days, and then they were differentiated to develop myotubes and used for further experiments. Briefly, muscle cells were exposed to either vehicle (10 µl of 95% ethanol) only (control, in 95% air and 5% CO2), DHEA (Wako Pure Chemical Industries, Osaka, Japan), or testosterone (Sigma, St. Louis, MO) for 24 h, and then cells were collected for analysis. DHEA and testosterone were dissolved in 95% ethanol. To make each concentration (50, 100, and 300 µM) of DHEA and testosterone, the adjusted volume of DHEA and testosterone was inserted into 10 µl of ethanol. The final incubation volume was all 2-ml medium, including 10-µl ethanol with DHEA or testosterone. The following doses were used for both DHEA and testosterone: 50, 100, and 300 µM. The 5{alpha}-reductase inhibitor (Sigma, Finasteride, Steinheim, Germany) was dissolved in 5 µl of 95% ethanol to a concentration of 10–9–10–5 M, which corresponded to 300 µM concentration of DHEA and testosterone. A 5{alpha}-reductase inhibitor was added for 1 h before testosterone or DHEA addition after 7 days of incubation. After treatment of DHEA and testosterone, medium was completely removed, and then cell samples were washed two times with PBS and harvested with 500 µl homogenate buffer containing 25 mM Tris·HCl, pH 7.6, 150 mM NaCl, 10% Nonidet P-40, 10% sodium deoxycholate, 0.1% sodium lauryl sulfate (SDS), and phenylmethylsulfonyl fluoride. Samples were rotated for 30 min at 4°C and then centrifuged at 10,000 g for 30 min. The supernatant was collected and stored at –80°C to analyze the gene expression of 3β-HSD, 17β-HSD, and 5{alpha}-reductase types 1 and 2, intracellular levels of DHT, protein expression of GLUT-4 and GLUT-4 translocation from cell cytoplasm to cell membrane, total and phosphorylated Akt, phosphorylated PKC-{zeta}/{lambda}, and enzyme activities of PFK and HK. All of the above and subsequent experimental protocols in the present study were approved by the Committee on Animal Research at the University of Tsukuba.

RT-PCR. Total tissue RNA was isolated using Isogen (Nippon Gene, Toyama, Japan), according to previous studies (1, 16). Total tissue RNA (2 µg) was primed with 0.05 µg of oligo d(pT)12–18 and reverse transcribed by omniscript reverse transcriptase using a cDNA synthesis kit (Qiagen, Tokyo, Japan). The reaction was performed at 37°C for 60 min.

PCR was performed according to the previous report (15) with minor modifications. Each PCR reaction contained 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 mM dNTP, 0.5 µM gene-specific primers, and 0.025 U/µl ExTq polymerase (Takara, Shiga, Japan). The gene-specific primers were synthesized according to the published cDNA sequences for each of the following mRNA: 3β-HSD (31), 17β-HSD type 1 (12), 5{alpha}-reductase type 1 (11) and type 2 (3), and β-actin (25).

The sequences of the oligonucleotides were as follows: 3β- HSD forward: 5'-AGCCAATCCAGTGTATGTAGGC-3', 3β-HSD reverse: 5'-AGGTTTTCTGCTTGGCTTCCTC-3'; 17β-HSD forward: 5'-AAGTTTGCGCTCGAAGGTCTGC-3', 17β-HSD reverse: 5'-TGCAGACCATCCCAATTCAGCT-3'; 5{alpha}-reductase type 1 forward: 5'-CCTGCTGGCTATGTTTCTGATCCA-3', 5{alpha}-reductase type 1 reverse: 5'-TATCACCATGCCCACTAACCACAG-3'; 5{alpha}-reductase type 2 forward: 5'-TGCTGGCTCTCTTCTCTGCACATT-3', 5{alpha}-reductase type 2 reverse: 5'-TGTGAAAAAGGCGAAAGCGAAGGC-3'; β-actin forward: 5'-GAAGATCCTGACCGAGCGTA-3', β-actin reverse: 5'-CGTACTCCTGCTTGCTGATCC-3'.

PCR was carried out using a PCR thermal cycler (PCR system 9700, Applied Biosystems). The cycle profiling included denaturation for 30 s at 94°C, annealing for 30 s at 60°C for 3β-HSD and 17β-HSD, 42°C for 5{alpha}-reductase, and extension for 60 s at 72°C. The amplified PCR products were electrophoresed on 1.2% agarose gels, stained with ethidium bromide, visualized by an ultraviolet transilluminator, and photographed by M085D (Polaroid, Waltham, MA).

Sandwich-enzyme immunoassay. Intracellular levels of DHT in skeletal muscle extracts were determined using a sandwich-enzyme immunoassay kit (IBL, Humburg, Germany). All techniques and materials used in these analyses were in accordance with the manufacturer's protocol. The immobilized antibodies were polyclonal raised against DHT, whereas the secondary horseradish-peroxidase-coupled antibody was monoclonal. Optical density at 450 nm was qualified on a microplate reader using BioLumin 960 (Molecular Dynamics, Tokyo, Japan). All samples were assayed in duplicate.

Immunoblot analysis. Cultured cells were homogenized with 20 mM Tris·HCl (pH 7.8), 300 mM NaCl, 2 mM EDTA, 2 mM DTT, 2% Nonidet P-40, 0.2% SDS, 0.2% sodium deoxycholate, 0.5 mM phenylmethylsulfonyl fluoride, 60 µg/ml aprotinin, and 1 µg/ml leupeptin. The homogenates were softly rotated for 30 min at 4°C and centrifuged at 12,000 g for 15 min at 4°C, and then the protein concentration of the resulting supernatant was determined. The samples of 10-µg protein were subjected to heat denaturation at 96°C for 7 min with Laemmli buffer. Protein concentrations were determined by the bicinchoninic acid protein assay reagents (Pierce, Rockford, IL) with bovine serum albumin as a standard. Western blot analyses of GLUT-4, phosphorylated Akt, total Akt, and phosphorylated PKC-{zeta}/{lambda} proteins were performed according to previous reports with minor modification (16, 26). Western blot analysis of GLUT-4 was performed by total protein fraction. Briefly, each sample was separated on SDS-PAGE, transferred on 10% gels, and then transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan) at 15 V for 60 min. The membrane was treated with blocking buffer 5% skim milk in phosphate-buffered saline contained 0.1% Tween 20 for 24 h at 4°C. The membrane was proved with polyclonal GLUT-4 (1:1,000 dilution with the blocking buffer; Chemicon International), serine 473-phosphorylated Akt 1:1,000 dilution with blocking buffer (Cell Signaling, Beverly, MA), Akt antibody 1:1,000 dilution with blocking buffer (Cell Signaling), and threonine 410/403-phosphorylated PKC-{zeta}/{lambda} 1:1,000 dilution with blocking buffer (Cell Signaling), for 1 h at room temperature. The membrane was washed three times and then incubated with a horseradish peroxidase-conjugated secondary antibody, anti-rabbit immunoglobulin, 1:3,000 dilution, with blocking buffer (GE Healthcare Biosciences, Piscataway, NJ, and Cell Signaling), for 1 h at room temperature. The membrane was then washed with PBS-Tween 20 three times. Finally, GLUT-4, phosphorylated Akt, total Akt, and phosphorylated PKC-{zeta}/{lambda} proteins were detected by an enhanced chemiluminescence plus system (GE Healthcare Biosciences) and exposed to a hyper film (GE Healthcare Biosciences). The immunoblots were scanned with a charge-coupled device camera. The density of the blots was quantified by using a densitometry system (Bio-Rad, Quantity One, version 4.4). Values are expressed as means ± SE of four independent preparations of cells, and each was performed in triplicate.

Preparation of the cytosolic and plasma membrane protein fraction. For determination of GLUT-4 translocation, two different membrane fractions were used, according to Kristiansen et al. (20) and Benomar et al. (6). Briefly, the cells were scraped in buffer A containing 20 mM Tris, pH 7.4, 1 mM EDTA, 0.25 mM EGTA, 0.25 M sucrose, 1 mM DTT, 50 mM NaF, 25 mM sodium pyrophosphate, and 40 mM β-glycerophosphate. The resulting homogenates were clarified 400 g for 15 min. The supernatant was centrifuged at 50,000 rpm for 1 h. The resulting pellet was homogenized in buffer A and sampled as a cytosol protein fraction. Proteins from different fractions were solubilized for 1 h at room temperature in buffer B containing 20 mM Tris, pH 7.4, 1 mM EDTA, 0.25 mM EGTA, 2% Triton X-100, 50 mM NaF, 25 µM sodium pyrophosphate, and 40 mM β-glycerophosphate. The homogenate was centrifuged, and the supernatant was spun for 1 h at 55,000 rpm and then sampled as a plasma membrane fraction. GLUT-4 protein level was measured in both plasma cytosol and membrane fractions, and translocation was evaluated by the difference in protein levels in cytosol and membrane fractions.

Enzyme activities. To determine enzyme activity of PFK, 10 µl of each extracted sample, with 1.5 µl of 1 M potassium fluoride added, were incubated for 5 min at 30°C in a 190-µl incubation mixture containing 25 mM β-glycerophosphate, 100 µM DTT, 12 mM glycylglycine, 0.2 mM fructose 6-phosphate, 0.5 mM ATP, and 0.1 mM β-NADH. The reaction was determined spectrophotometrically at 340 nm for 5 min (19).

To determine enzyme activity of HK, 10 µl of each sample were incubated for 5 min at 30°C in a 150-µl incubation mixture containing 125 µM Tris·HCl (pH 7.6) and 0.6 units of glucose 6-phosphate dehydrogenase (Sigma-Aldrich, Tokyo, Japan). The reaction was initiated by the addition of 40 µl of 100 mM D(+)-glucose and was then determined spectrophotomecally at 340 nm for 5 min.

Statistical analysis. All values are expressed as the means ± SE. Statistical evaluation of the data was by one-way ANOVA. When analysis revealed significant differences, a post hoc comparison test was used to correct for multiple comparisons (Bonferroni/Dunn test). P < 0.05 was considered significant for ANOVA and P < 0.01 for post hoc test.


   RESULTS
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DISCUSSION
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3β-HSD and 17β-HSD type 1 gene expression and testosterone concentration. We confirmed the existence of 3β-HSD and 17β-HSD type 1 mRNAs in cultured skeletal muscle in the present study and detected the gene expression of 3β-HSD and 17β-HSD type 1 in skeletal muscle (Fig. 1A). Additionally, intracellular testosterone level in the skeletal muscle was significantly increased by the addition of 300 µM DHEA (Fig. 1B).


Figure 1
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  		Fig. 1. mRNA expressions of 3β-hydroxysteroid dehydrogenase (HSD) and 17β-HSD (A) and changes in intracellular testosterone concentration with dehydroepiandrosterone (DHEA) addition to cultured skeletal muscle cells (B). A: representative images of mRNA expressions of 3β-HSD and 17β-HSD type 1 revealed by RT-PCR. The mRNA of the rat ovary was used as a positive control, and β-actin mRNA was used as an internal control. B: changes in intramuscular levels of testosterone in skeletal muscle with DHEA addition. Values are means ± SE of 3 independent measurements. #P < 0.01 compared with the control by the post hoc test.

 
5{alpha}-Reductase type 1 and 2 gene expressions and 5{alpha}-DHT concentration. To investigate whether 5{alpha}-reductase type 1 and type 2 exist in the skeletal muscle, 5{alpha}-reductase gene expression was examined by RT-PCR. The gene expressions of 5{alpha}-reductase type 1 and type 2 were detected in the cultured skeletal muscle (Fig. 2A).


Figure 2
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  		Fig. 2. 5{alpha}-Reductase type 1 and type 2 mRNA expressions (A) and changes in 5{alpha}-dihydrotestosterone (DHT) concentration with addition of testosterone or DHEA to cultured skeletal muscle (B). A: representative images of expressions of 5{alpha}-reductase type 1 and type 2 mRNA revealed by RT-PCR. The mRNA of the rat ovary was used as a positive control, and β-actin mRNA was used as an internal control. B: changes in intramuscular concentration of DHT with testosterone or DHEA addition. Values are means ± SE of 3 independent measurements. #P < 0.01 compared with the control.

 
Furthermore, we investigated whether skeletal muscle is capable of using testosterone or DHEA as a precursor for local DHT production, by adding testosterone or DHEA to cultured skeletal muscle. Intracellular levels of DHT in skeletal muscle increased in a dose-dependent manner with the addition of testosterone or 50–300 µM DHEA. There were significant increases in DHT concentration with 100 and 300 µM testosterone addition compared with the control (Fig. 2B). In the DHEA addition, only a 300 µM dose had a significant effect on the increase in DHT concentration compared with the control (Fig. 2B).

GLUT-4 protein expression and translocation. There were significant increases in GLUT-4 protein expression with 100 and 300 µM testosterone addition compared with the control (Fig. 3A), whereas there was a significant increase with only 300 µM DHEA addition (Fig. 3A).


Figure 3
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  		Fig. 3. Glucose transporter-4 (GLUT-4) protein expression (A) and its translocation with addition of testosterone or DHEA to cultured skeletal muscle (B). A, top: representative images of immunoblotting for GLUT-4 protein expression. Arrows indicate immunoblot bands for GLUT-4 protein. A, bottom: results of statistical analyses for levels of GLUT-4 protein expression by densitometer. #Significant increase (P < 0.0125) compared with the control. B, top: representative images of immunoblotting for GLUT-4 proteins in cytosol and the membrane. Arrows indicate immunoblot bands for GLUT-4 protein. B, bottom, left: results of statistical analyses for levels of GLUT-4 protein expression by densitometer. B, bottom, right: difference in GLUT-4 protein levels in cytosol and membrane fractions as the level of GLUT-4 translocation. Values are means ± SE of 3 independent measurements. #P < 0.01 compared with the control. D Con, DHEA control; T Con, testosterone control; D 300, 300 µM DHEA addition; T 300, 300 µM testosterone addition; AU, arbitrary unit.

 
Both 300 µM testosterone and DHEA additions significantly increased GLUT-4 protein levels in cytosol (Fig. 3B) and in the plasma membrane (Fig. 3B). The difference in cytosol and membrane GLUT-4 protein levels was significantly greater with a 300 µM dose of testosterone and DHEA than the control (Fig. 3B).

Akt and PKC-{zeta}/{lambda} phosphorylation. Akt phosphorylation was evaluated by the ratio of phosphorylated level to total levels in the present study. Akt phosphorylation increased in a dose-dependent manner by the addition of testosterone or 50–300 µM DHEA (Fig. 4A). There were significant increases in Akt phosphorylation with 50, 100, and 300 µM doses of testosterone addition (Fig. 4A). For DHEA addition, there was a significant increase with only 300 µM dose addition (Fig. 4A).


Figure 4
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  		Fig. 4. Akt and protein kinase C (PKC)-{zeta}/{lambda} phosphorylations with addition of testosterone or DHEA to cultured skeletal muscle. A, top: representative images of immunoblotting for phosphorylated Akt (p-Akt) and total Akt protein. Arrows indicate immunoblot bands for each protein. A, bottom: results of statistical analyses for the ratio of p-Akt protein and total Akt protein expression. The ratio of Akt phosphorylation in skeletal muscle was calculated by dividing total Akt protein level by p-Akt protein level. B, top: representative images of immunoblotting for phosphorylated (Phospho)-PKC-{zeta}/{lambda} protein. Arrow indicates immunoblot bands for PKC-{zeta}/{lambda} protein. B, bottom: results of statistical analyses for levels of phosphorylated PKC-{zeta}/{lambda} expression. Values are means ± SE of 3 independent measurements. #P < 0.01 compared with the control.

 
Phosphorylated PKC-{zeta}/{lambda} significantly increased with both 300 µM dose addition of testosterone and DHEA compared with the control (Fig. 4B).

Effects of a DHT inhibitor on GLUT-4 protein expression and Akt and PKC-{zeta}/{lambda} phosphorylation. With the DHT inhibitor addition, GLUT-4 protein expressions were significantly decreased with both 300 µM testosterone and DHEA additions compared with the control (Fig. 5A).


Figure 5
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  		Fig. 5. Effects of a DHT inhibitor on GLUT-4 protein expression, Akt, and PKC-{zeta}/{lambda} phophorylations, with addition of testosterone or DHEA to cultured skeletal muscle. A, top: representative images of immunoblotting for GLUT-4 protein expression, with and without a DHT inhibitor. Arrow indicates the immunoblot band for the protein. A, bottom: results of statistical analyses for levels of GLUT-4 protein expression. Representative images of immunoblotting for p-Akt and total Akt (B, top) and phosphorylated PKC-{zeta}/{lambda} expression (C, top), with and without a DHT inhibitor, are shown. Arrows indicate immunoblot bands for the proteins. B and C, bottom: results of statistical analysis for the ratio of p-Akt to total Akt (B) and the phosphorylated PKC-{zeta}/{lambda} (C) expression. Values are means ± SE of 3 independent measurements. #P < 0.01 compared with the control. {dagger}P < 0.05 compared with inhibitor addition.

 
Both Akt and PKC-{zeta}/{lambda} phosphorylations were significantly decreased by the DHT inhibitor addition (Fig. 5, B and C).

Glycolytic enzyme activities. To investigate whether testosterone or DHEA has the capacity to enhance glucose metabolic activity, especially glycolysis, activities of HK and PFK, which are the main enzymes in the glycolytic pathway, were examined. HK activity increased significantly with both 100 and 300 µM addition of testosterone or 300 µM addition of DHEA compared with the control (Fig. 6A). PFK activity increased significantly with both 100 and 300 µM additions of testosterone and 300 µM addition of DHEA (Fig. 6B).


Figure 6
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  		Fig. 6. Hexokinase (HK; A) and phosphofructokinase (PFK; B) enzyme activities with addition of testosterone or DHEA to cultured skeletal muscle. Values are means ± SE of 3 independent measurements. #P < 0.01 compared with the control.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main new findings of the present study were that local production of DHT increased with the addition of testosterone or DHEA and the gene expressions of 5{alpha}-reductase type 1 and type 2 were detected in cultured skeletal muscle, suggesting that skeletal muscle is capable of locally synthesizing DHT from testosterone or DHEA. Second, the addition of testosterone and DHEA to cultured skeletal muscle induced the elevation of GLUT-4 protein expression and also accelerated its translocation from cytosol to the membrane. The addition of testosterone and DHEA to cultured skeletal muscle enhanced both Akt and PKC-{zeta}/{lambda} phosphorylations, which have critical roles in GLUT-4-regulated signaling pathways. Moreover, GLUT-4 protein expression and Akt and PKC-{zeta}/{lambda} phosphorylations were significantly blocked with DHT inhibitor addition. Thus locally synthesized testosterone or DHT affects the expression and translocation of GLUT-4 via Akt and PKC-{zeta}/{lambda} pathways in skeletal muscle. Furthermore, the present study showed the enhancement of main enzyme activities (HK and PFK) in the glycolytic pathway by testosterone and DHEA additions. HK and PFK activities are enhanced by GLUT-4 overexpression in skeletal muscle (21); therefore, the upregulation of GLUT-4 may result in the enhancement of enzyme activities contributing to glycolysis in skeletal muscle.

The 5{alpha}-reductase type 1 and type 2 enzymes, which convert enzymes from testosterone to DHT, are expressed in various tissues, i.e., the ovary, liver, skin, and prostate (4, 29). Recently, our laboratory demonstrated the presence of local mRNA and protein expressions of 3β-HSD and 17β-HSD, which are important converting enzymes from DHEA to testosterone in the skeletal muscle, and they resulted in activation of the related metabolic pathway (1). In the present study, we again detected the gene expressions of 3β-HSD and 17β-HSD and, for the first time, detected those of 5{alpha}-reductase type 1 and type 2 in cultured skeletal muscle. Interestingly, the addition of not only testosterone, but also DHEA, induced the synthesis of DHT in the skeletal muscle. Therefore, it is considered that testosterone is synthesized from DHEA through 3β-HSD and 17β-HSD enzymes, and DHT is subsequently synthesized form testosterone through 5{alpha}-reductase enzymes. Thus skeletal muscle may be local steroidogenesis tissue, metabolizing bioactive steroid hormones. However, we could not provide more direct evidences using radiolabeled DHEA and testosterone or elimination of several enzymes antisense. Therefore, further study needs to clarify whether skeletal muscle can synthesize DHT from DHEA.

It is well known that insulin upregulates Akt phosphorylation via PI 3-kinase, resulting in an increase in translocation and utilization with GLUT-4 expression in skeletal muscle (2). Bjprnholm et al. (7) reported that both insulin-stimulated PI 3-kinase and Akt activations decreased and subsequently GLUT-4 expression decreased in skeletal muscle in type 2 diabetic patients. Recently, Kang et al. (18) reported that testosterone and its metabolite DHT induced the activation of Akt through the upregulation of PI 3-kinase in osteoblast cells, and they also demonstrated that the inhibition of PI 3-kinase abolished DHT-induced Akt phosphorylation (18). Additionally, for PKC-{zeta}/{lambda} phosphorylation, Campbell et al. (9) reported that DHEA enhanced insulin-stimulated PKC-{zeta}/{lambda} activation, and this activation may modulate GLUT-4 translocation in skeletal muscles. Our results suggested that the increase in GLUT-4 protein expression and Akt and PKC-{zeta}/{lambda} phosphorylations were induced, corresponding to the increase in intramuscular DHT level in skeletal muscle with testosterone or DHEA addition. Moreover, a DHT inhibitor blocked the effects of testosterone or DHEA addition on Akt-GLUT-4 and PKC-{zeta}/{lambda}-GLUT-4 pathways. Subsequently, we confirmed that the glucose metabolism-related signaling pathway is literally activated by androgenic hormones. Taken together, testosterone or DHT-induced increase in the expression of GLUT-4 protein and the concomitant enhancement of translocation via the activation of Akt and PKC-{zeta}/{lambda} may result in increased glucose uptake and utilization in skeletal muscle. Although both testosterone and DHEA enhanced GLUT-4 translocation, DHEA-induced GLUT-4 translocation was less than that of testosterone induced (vs. DHEA, 2.6-fold). According to the results of GLUT-4 protein expression and Akt phosphorylation, these activations occurred in a lower dose of testosterone compared with DHEA dose. Thus these testosterone responses were more sensitive than DHEA. DHEA is metabolized to DHT through 3β-HSD, 17β-HSD, and 5{alpha}-reductase enzymes, whereas testosterone is metabolized to DHT only through 5{alpha}-reductase enzymes. These different metabolizing pathways may be a causal factor of the different sensitivity of glucose signaling pathway activation between testosterone and DHEA.

DHEA and its sulfate-bound form (DHEAS) are important precursors of sex steroid hormones in target tissues (5). In the present study, DHEA, as well as testosterone, affected the signaling pathway involved in the regulation of glucose metabolism in skeletal muscle; hence this suggests that skeletal muscle is capable of the uptake and utilization of DHEA, a precursor of testosterone. Interestingly, plasma DHEAS levels in humans are 100–500 times higher than testosterone (23), thus suggesting that circulating DHEAS and testosterone are important substrates or precursors in terms of the local regulation of glucose metabolism in skeletal muscle.

Since diabetes is one of the risk factors of coronary artery disease, hypertension, and hyperlipidemia, it is crucially important to prevent, and treat patients with, hyperglycemia. To improve insulin resistance, insulin administration or exercise is a therapeutic strategy, because these therapies increase glucose utilization in skeletal muscle. The present study shows that the glucose metabolism-related signaling pathway and enzyme activities were enhanced with DHEA or testosterone addition to skeletal muscle. The decline in circulating DHEAS levels with aging and/or obesity has been correlated to the gradually increasing prevalence of diabetes (24). Thus sex steroid hormones are possible new therapeutic and prevention candidates to restore impaired insulin signal transduction in skeletal muscle for patients with hyperglycemia, which needs to be the focus of further studies. Although the present study was conducted to confirm that DHEA and/or testosterone stimulates glucose signaling pathway in skeletal muscle cell, the concentration of DHEA and testosterone in the present study was more than 50–100 times higher than healthy human blood concentration (20–30 µM). The further study should focus on the effect of the physiological range of DHEA administration in vivo. Moreover, it needs to confirm whether the DHEA administration affects the glucose-signaling pathway in skeletal muscle.

In conclusion, the present study found the gene expression of 5{alpha}-reductase type 1 and type 2 and intramuscular production of DHT, with the addition of testosterone or DHEA in skeletal muscle, suggesting that skeletal muscle may be capable of locally synthesizing DHT from testosterone or DHEA. Importantly, the addition of testosterone or DHEA to cultured skeletal muscle induced an elevation of GLUT-4 expression and translocation with increased Akt and PKC-{zeta}/{lambda} phosphorylations, but these alterations were blocked with DHT inhibitor addition. Moreover, the main glycolytic enzyme activities were enhanced with the addition of testosterone or DHEA to skeletal muscle. Thus locally synthesized DHT may affect the activation of glucose metabolism-regulated intracellular signaling pathway in skeletal muscle.


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This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17300204).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Ajisaka, Graduate School of Comprehensive Human Sciences, Univ. of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan (e-mail: ajisakas{at}taiiku.tsukuba.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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