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Search for other papers by Pierre-Gilles Blanchard in
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Search for other papers by Van Luu-The in
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Recently, we have shown that human and monkey type 12 17β-hydroxysteroid dehydrogenases (17β-HSD12) are estrogen-specific enzymes catalyzing the transformation of estrone (E1) into estradiol (E2). To further characterize this novel steroidogenic enzyme in an animal model, we have isolated a cDNA fragment encoding mouse 17β-HSD12 and characterized its enzymatic activity. Using human embryonic kidney cells (HEK)-293 cells stably expressing mouse 17β-HSD12, we found that in contrast with the human and monkey enzymes, which are specific for the transformation of E1 to E2, mouse 17β-HSD12 also catalyzes the transformation of 4-androstenedione into testosterone (T), dehydroepiandroster-one (DHEA) into 5-androstene-3β,17β-diol (5-diol), as well as androsterone into 5α-androstane-3α,17β-diol (3α-diol). Previously, we have shown that the specificity of human and monkey 17β-HSD12s for C18-steroid is due to the presence of a bulky phenylalanine (F) at position 234 creating steric hindrance, preventing the entrance of C19-steroids into the active site. To determine whether the smaller size of the corresponding leucine (L) in the mouse sequence is responsible for the entrance of androgenic substrates, we performed site-directed mutagenesis to substitute Leu 234 for Phe in the mouse enzyme. In agreement with our hypothesis, the mutated enzyme has a highly reduced ability to metabolize androgens. mRNA quantification in several mouse tissues using real-time PCR shows that mouse 17β-HSD12 mRNA is highly expressed in the female clitoral gland, male preputial gland, as well as in retroperitoneal fat and adrenal of both sexes. The differential androgenic/estrogenic substrate specificity of type 12 17β-HSD in the mouse and primates seems to agree with the observation that androgen and estrogen in the mouse are provided almost exclusively by gonads, while in primates an important part of these steroid hormones are produced locally from adrenal precursors.
Search for other papers by Serge Desnoyers in
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Search for other papers by Pierre-Gilles Blanchard in
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Search for other papers by David L Baillie in
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Search for other papers by Van Luu-The in
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Mutations that inactivate LET-767 are shown to affect growth, reproduction, and development in Caenorhabditis elegans. Sequence analysis indicates that LET-767 shares the highest homology with human types 3 and 12 17β-hydroxysteroid dehydrogenases (17β-HSD3 and 12). Using LET-767 transiently transfected into human embryonic kidney-293 cells, we have found that the enzyme catalyzes the transformation of both 4-androstenedione into testosterone and estrone into estradiol, similar to that of mouse 17β-HSD12 but different from human and primate enzymes that catalyze the transformation of estrone into estradiol. Previously, we have shown that amino acid F234 in human 17β-HSD12 is responsible for the selectivity of the enzyme toward estrogens. To assess whether this amino acid position 234 in LET-767 could play a role in androgen–estrogen selectivity, we have changed the methionine M234 in LET-767 into F. The results show that the M234F change causes the loss of the ability to transform androstenedione into testosterone, while conserving the ability to transform estrone into estradiol, thus confirming the role of amino acid position 234 in substrate selectivity. To further analyze the structure–function relationship of this enzyme, we have changed the three amino acids corresponding to lethal mutations in let-767 gene. The data show that these mutations strongly affect the ability of LET-767 to convert estrone in to estradiol and abolish its ability to transform androstenedione into testosterone. The high conservation of the active site and amino acids responsible for enzymatic activity and substrate selectivity strongly suggests that LET-767 shares a common ancestor with human 17β-HSD3 and 12.
Search for other papers by Fernand Labrie in
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Search for other papers by Van Luu-The in
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Search for other papers by Ezequiel Calvo in
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Search for other papers by Céline Martel in
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Search for other papers by Marie-Hélène Lévesque in
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Search for other papers by Claude Labrie in
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Tetrahydrogestrinone (THG) is a recently identified compound having the greatest impact in the world of sports. In order to obtain a highly accurate and sensitive assessment of the potential anabolic/androgenic activity of THG, we have used microarrays to identify its effect on the expression of practically all the 30 000 genes in the mouse genome and compared it with the effect of dihydrotestosterone (DHT), the most potent natural androgen. Quite remarkably, we found that 671 of the genes modulated by THG in the mouse muscle levator ani are modulated in a similar fashion by DHT, while in the gastrocnemius muscle and prostate, 95 and 939 genes respectively, are modulated in common by the two steroids. On the other hand, THG is more potent than DHT in binding to the androgen receptor, while, under in vivo conditions, THG possesses 20% of the potency of DHT in stimulating prostate, seminal vesicle and levator ani muscle weight in the mouse. The present microarray data provide an extremely precise and unquestionable signature of the androgenic/anabolic activity of THG, an approach which should apply to the analysis of the activity of any anabolic steroid.
Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
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Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
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Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
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Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
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Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
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Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
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Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
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Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
Search for other papers by Van Luu-The in
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Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Québec, Québec, Canada G1V 4G2
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We examined 5α-dihydrotestosterone (5α-DHT) inactivation and the expression of several steroid-converting enzymes with a focus on aldoketoreductases 1C (AKR1C), especially AKR1C2, in abdominal adipose tissue in men. AKR1C2 is mainly involved in the conversion of the potent androgen 5α-DHT to its inactive forms 5α-androstane-3α/β,17β-diol (3α/β-diol). Subcutaneous (s.c.) and omental (Om) adipose tissue biopsies were obtained from 21 morbidly obese men undergoing biliopancreatic derivation surgery and 11 lean to obese men undergoing general abdominal surgery. AKR1C2 mRNA and 5α-DHT inactivation were detected in both s.c. and Om adipose tissue. After incubation of preadipocytes with 5α-DHT, both 3α-diol and 3β-diol were produced through 3α/β-ketosteroid reductase (3α/β-HSD) activity. In preadipocyte cultures, 3α-reductase activity was significantly predominant over 3β-reductase activity in cells from both the s.c. and Om compartments. Expression levels of AKR1C1, AKR1C3 and of the androgen receptor were significantly higher in s.c. versus Om adipose tissue while mRNA levels of 17β-HSD-2 (hydroxysteroid dehydrogenase type 2) and 3(α→β)-hydroxysteroid epimerase were significantly higher in Om fat. 3α/β-HSD activity was mainly detected in the cytosolic fraction, suggesting that AKR1C may be responsible for this reaction. Experiments with isoform-specific AKR1C inhibitors in preadipocytes showed that AKR1C2 inhibition significantly decreased 3α-HSD and 3β-HSD activities (3α-HSD: 30 ± 24% of control for s.c. and 32 ± 9% of control for Om, 3β-HSD: 44 ± 12% of control for s.c.). When cells were incubated with both AKR1C2 and AKR1C3 inhibitors, no significant additional inhibition was observed. 5α-DHT inactivation was significantly higher in mature adipocytes compared with preadipocyte cultures in s.c. adipose tissue, as expressed per microgram total protein (755 ± 830 versus 245 ± 151 fmol 3α/β-diol per μg protein over 24 h, P < 0.05 n = 10 cultures). 5α-DHT inactivation measured in tissue homogenates was significantly higher in the s.c. depot compared with Om fat (117 ± 39 versus 79 ± 38 fmol 3α/β-diol per μg prot over 24 h, P < 0.0001). On the other hand, Om 3α/β-HSD activity was significantly higher in obese men (body mass index (BMI) ≥ 30 kg/m2) compared with lean and overweight men (84 ± 37 versus 52 ± 30 fmol 3α/β-diol per μg protein over 24 h, P < 0.03). No difference was found in s.c. 3α/β-HSD activity between these groups. Positive correlations were found between s.c. 5α-DHT inactivation rate and circulating levels of the androgen metabolites androsterone-glucuronide (r = 0.41, P < 0.02) and 3α-diol-glucuronide (r = 0.38, P < 0.03) and with the adrenal precursor androstenedione (r = 0.42, P < 0.02). In conclusion, androgen inactivation was detected in abdominal adipose tissue in men, with higher 3α/β-HSD activity in the s.c. versus Om depot. Higher Om 5α-DHT inactivation rates were found in obese compared with lean men. Further studies are required to elucidate whether local androgen inactivation in abdominal adipose tissue is involved in the modulation of adipocyte metabolism and regional fat distribution in men.