Steroid-converting enzymes in human adipose tissues and fat deposition with a focus on AKR1C enzymes.

Adipocytes express various enzymes, such as aldo-keto reductases (AKR1C), 11ß-hydroxysteroid dehydrogenase (11ß-HSD), aromatase, 5α-reductases, 3ß-HSD, and 17ß-HSDs involved in steroid hormone metabolism in adipose tissues. Increased activity of AKR1C enzymes and their expression in mature adipocytes might indicate the association of these enzymes with subcutaneous adipose tissue deposition. The inactivation of androgens by AKR1C enzymes increases adipogenesis and fat mass, particularly subcutaneous fat. AKR1C also causes reduction of estrone, a weak estrogen, to produce 17ß-estradiol, a potent estrogen and, in addition, it plays a role in progesterone metabolism. Functional impairments of adipose tissue and imbalance of steroid biosynthesis could lead to metabolic disturbances. In this review, we will focus on the enzymes involved in steroid metabolism and fat tissue deposition.

The organelles containing porcine 17 beta-estradiol dehydrogenase are peroxisomes.

Porcine 17 beta-estradiol dehydrogenase was recently purified and cloned. It catalyzes the NAD(+)-dependent oxidation of estradiol to estrone 360-fold more efficiently than the reverse reaction with NADPH. Immunogold electron microscopy localizes 17 beta-estradiol dehydrogenase in organelles of 120 to 500 nm with moderate electron-dense matrices bounded by single membranes. Antibodies against the peroxisomal markers catalase and acyl-CoA oxidase recognize the same organelles in double-labeling studies. This is the first report on the participation of peroxisomes in the metabolism of estradiol.

Alterations in the subcellular distribution of 17 beta-estradiol dehydrogenase in porcine endometrial cells over the course of the estrous cycle.

The uteri of German landrace gilts slaughtered at different days of the cycle were processed for immunocytochemistry and biochemical analyses. Plasma was collected for hormone assays. The monoclonal antibody F1 against the structure-bound 17 beta-estradiol dehydrogenase of porcine endometrial epithelium was applied to rehydrated paraffin sections either as a direct, peroxidase-linked probe or in combination with a fluorescing secondary antibody. The oxidation of estradiol was measured in homogenates of tissue powdered in liquid nitrogen. Immunoreactivity was restricted to endometrial epithelium. In the glandular epithelium, faint dots of fluorescence became visible at day 4, which apparently coalesced to spherical structures of 2-4 microns diameter at the cell basis between days 11 through 17 before disappearing by day 18. A similar distribution was observed for the oxidation products of diaminobenzidine beginning with a faint uniform staining and followed by the appearance of intensely stained basal bodies persisting until day 17. Essentially the same time course was seen in the luminal epithelium but with a different distribution. Immunoreactive material amassed in the apical region of the cells, but the conspicuous aggregations were absent. Time course and intensities of the immunological responses are matched by the enzymatic activity measured in parallel. Both correlate with the plasma progesterone levels, suggesting an induction of the enzyme by the hormone. An involvement of the cytoskeleton in the sequence of subcellular distribution patterns is discussed.

The 17 beta-oestradiol dehydrogenase of pig endometrial cells is localized in specialized vesicles.

Two monoclonal antibodies against the 17 beta-oestradiol dehydrogenase of pig endometrial cells have been used in localization studies with immunogold electron microscopy. The antibodies attach both to a fraction of dehydrogenase-rich cytoplasmic vesicles isolated from homogenates and to vesicles of similar appearance in cells. The vesicles are filled with electron-dense material. Their tagging intensity indicates a high degree of specialization. Endometrial cells from mature animals contain a host of dehydrogenase vesicles, and cells from prepubertal animals only a few. Functional aspects of the novel organelle are discussed.

The histochemistry of isocitric and oestradiol-17 beta dehydrogenases in the endometrium of postmenopausal women treated with oestrogens and progestogens.

Endometrium was obtained from postmenopausal women during treatment with either oestrogen alone or on the third, sixth, tenth or twelfth day of combined therapy with oestrogens and progestogens. The activities of oestradiol-17 beta and isocitric dehydrogenases were measured in homogenates of the whole tissue and the enzymes were also located histochemically. Oestradiol dehydrogenase was located exclusively within the epithelium, whilst isocitric dehydrogenase was found in both epithelium and stroma, and also in stromal arterioles. Histochemically, oestradiol dehydrogenase was found in all the specimens which exhibited biochemical activity but in none of those from which it was absent. Isocitric dehydrogenase however, was seen in all tissue sections despite widely varying levels of biochemical activity in the homogenate. The method for measuring isocitric dehydrogenase activity was therefore nonspecific, whilst that for oestradiol dehydrogenase was reliable and low levels of enzyme activity could be detected. The latter technique may therefore be useful to predict the sensitivity of endometrial carcinomata to progestogens.

Quantitative determination of oestradiol-17 beta hydroxysteroid dehydrogenase: increased sensitivity by HPLC separation of the hormones permits the measurement of enzyme activity in cryostat sections.

The activity of the oestradiol-17 beta hydroxysteroid dehydrogenase in human endometrial and breast cancer specimens was determined by the NAD-dependent conversion of oestradiol-17 beta to oestrone. The sensitivity of the determination was improved by the separation of the hormones by HPLC. We are now able to determine oestradiol-17 beta hydroxysteroid dehydrogenase quantitatively in cryostat sections. A clear correlation of serum progesterone levels and oestradiol-17 beta hydroxysteroid dehydrogenase activity in the endometrium was demonstrated, and we found a more than 30-fold increase in enzyme activity after the progesterone surge. In contrast, in breast cancer samples, we found no correlation between oestradiol-17 beta hydroxysteroid dehydrogenase and the measured serum parameters.

Immunohistochemical localization of aromatase cytochrome P-450 and estradiol dehydrogenase in the syncytiotrophoblast of the human placenta.

Immunohistochemistry employing immunoglobulin G fractions raised against aromatase cytochrome P-450 and antiserum against 17 beta-estradiol dehydrogenase was used to localize these two steroid-converting enzymes in the human placenta. Immunostaining for both enzymes was found exclusively in the syncytiotrophoblast, while the underlying cytotrophoblast and the villus core did not stain. Ultrastructural examination of aromatase cytochrome P-450- and 17 beta-estradiol dehydrogenase-labeled sections disclosed immunoreactive product in the membranes of the endoplasmic reticulum; the nucleus, mitochondria, Golgi apparatus, and secretory granules were free of staining. These findings suggest that the syncytiotrophoblast is actively involved in the synthesis and metabolism of estrogens and in their role in placental endocrine function.

Assessment of the potency of orally administered progestins in women.

The effects of at least three doses of each of five orally administered progestins on estrogen-primed, postmenopausal endometrial biochemistry and morphologic features were analyzed. The progestins tested were norethindrone, medroxyprogesterone acetate (MPA), norgestrel, dydrogesterone, and progesterone. The dose required to elicit responses similar to those seen in premenopausal, secretory endometria was assessed for each of the parameters measured, and the relative potencies were calculated. Potencies, relative to a value of 1 for norethindrone, are L norgestrel 8 (D/L norgestrel 4), MPA 0.1, dydrogesterone 0.02, and progesterone 0.002. The dose of norethindrone required to elicit secretory phase activity was about 0.35 mg/day. These values agree with published data obtained with the use of different methods (delay of menstruation in premenopausal women, endometrial histologic features of estrogen-primed, ovariectomized women).

A comparison of the in vivo uptake and metabolism of 3H-oestrone and 3H-oestradiol by normal breast and breast tumour tissues in post-menopausal women.

After infusion of 3H-oestradiol or 3H-oestrone into post-menopausal women with breast cancer, there was a significant uptake of both steroids by breast tumour and normal tissue. The proportion of oestrogen present in tumour tissue as 3H-oestradiol after infusion of 3H-oestradiol (89.4 +/- 3.5%, mean +/- SD, n = 4) was significantly higher (p less than 0.001) than in normal breast tissue (72.8 +/- 3.3%) obtained from the same women. Similarly, after infusion of 3H-oestrone, the proportion of oestrogen present as 3H-oestradiol (48.4 +/- 14.4%) was significantly higher than in normal breast tissue (19.1 +/- 6.4%). These results suggest that conversion of oestrone to oestradiol is enhanced in breast tumour tissue with little metabolism of oestradiol. This would account for the higher concentrations of oestradiol reported in breast tumour tissue in the presence of increased oestradiol 17 beta-hydroxysteroid dehydrogenase activity.

The "dysharmonic luteal phase" syndrome: endometrial progesterone receptor and estradiol dehydrogenase.

The dysharmonic luteal phase (DLP) syndrome is defined by delayed endometrial maturation despite normal plasma progesterone (P) values. In ten patients with DLP the actual date of the endometrial biopsy, dated retrospectively, was 24.7 +/- 2.3 days, whereas the histologic date was 20.0 +/- 2.6 days. The concentration of cytosolic P receptor in DLP endometrium tended to be lower, whereas the concentration of nuclear receptor was significantly higher in DLP than in seven matched patients with normal luteal phases. Endometrial estradiol-dehydrogenase activities were identical in both groups. The DLP syndrome cannot be explained by a decreased sensitivity of the endometrium to P and is probably merely functional in nature.

Distribution of progesterone receptor, estradiol dehydrogenase, and 20 alpha-dihydroprogesterone dehydrogenase activities in human endometrial glands and stroma: progestin induction of steroid dehydrogenase activities in vitro is restricted to the glandular epithelium.

Endometrial glands were separated from stromal cells by collagenase digestion of human endometrium, and the distribution of progesterone (P) receptor and the activities of P-regulated enzymes, 17 beta-estradiol dehydrogenase (E2DH) and 20 alpha-dihydroprogesterone dehydrogenase (20 alpha DH), were determined in these preparations. Concentrations of cytosolic P receptor were estimated by Scatchard plot analysis of specific [3H]P binding in intact proliferative endometrium and in glands and stromal cells isolated from this tissue. Epithelial cells were more than 10-fold enriched in high affinity, P-specific binding sites compared to stromal cells. The binding constants (Kd) for [3H]P binding were essentially similar in undissociated endometrium, glandular epithelium, and stroma, ranging from 1--5 nM. The activities of E2DH and 20 alpha DH were also 3-fold higher in the glandular epithelium than in whole tissue or stroma during both proliferative and secretory stages of the menstrual cycle. In addition, the induction of these enzyme activities by progestin in vitro in cultured explants of proliferative endometrium was restricted to the glandular epithelium. Thus, the effects of P on E2DH and 20 alpha DH are expressed in the same cells that contain receptors for P.

Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17 beta-dehydrogenase.

Human placental estradiol 17 beta-dehydrogenase (E.C. 1.1.1.62) was inactivated at pH 6.3 by 3-bromo [2'-14C] acetoxy-1,3,5(10) estratrien-17-one, a know substrate. The affinity-alkylated enzyme was then hydrolyzed by trypsin. Radioactive peptides were initially isolated by gel filtration and identified according to which residue was alkylated. Tryptic peptides containing radioactive 3-carboxymethylhistidyl residues were further purified by cation-exchange chromatography. The population of these peptides varied, depending upon the conditions of enzyme inactivation. With 60 microM 3-bromo[2'-14]acetoxy-1,3,5 (10) estratrien -17-one four major peptides (a,b,c,d) each containing radioactive 3-carboxymethylhistidine, were eluted from the cation-exchange column. The alkylation of all of these peptides was completely suppressed when the enzyme was inactivated in the presence of excess estradiol-17 beta. The presence of equimolar NADPH during incubation greatly enhanced the alkylation of all four peptides. In the presence of NADPH, estradiol-17 beta most significantly decreased the formation of peptide d. Peptide d was the only peptide identified when the concentration of the alkylating steroid was lowered to 6 microM, a value approaching the Km. These observations indicate that peptide d is a histidyl-bearing peptide from the steroid-binding site which proximates the steroid A-ring. They further suggest that with the affinity labeling steroid at higher concentrations other nonspecific, hydrophobic sites on the enzyme are occupied and labeled.

Endometrial histology and biochemistry in climacteric women during oestrogen and oestrogen/progestogen therapy.

PIP: 177 patients were recruited between 1973 and 1977 to participate in a study to determine the endometrial response to exogenous estrogens and the modifying effect of progestogens on this response; hence, the endometrial proliferation during cyclical estrogen therapy and sequential estrogen/progestogen therapy was studied longitudinally. The patients were peri-or postmenopausal. Cyclical thereapy ranged from 2-47 months (mean, 15.1 months). With high-dose estrogen, cystic glandular hyperplasia was diagnosed in 16 (23%) patients, and atypical hyperplasia in 6 (9%); with low-dose regimens, cystic glandular hyperplasia was diagnosed in 4 (12%) and atypical hyperplasia in 2 (6%) patients. Sequentia therapy ranged from 2-50 months (mean, 16.2 months). With high dose regimens, 1 (2%) was found to have cystic glandular hyperplasia and 1 atient (2%) atypical hyperplasia; with low doses atypical hyperplasi was diagnosed in 1 patient (3%). these data therefore suggested that progestogens protected the endometrium against estrogen-induced stimulation, although complete protection was not afforded. Progestogens also depressed cytoplasmic progesterone receptor levels and induced the formation of estradiol 17-beta-dehydrogenase, as shown by the following findings: in all samples from Weeks 2 and 3, no activity of the enzyme was detectable. During norethisterone administration (Week 4), however, the activity rose to the premenopausal secretory range and was significantly higher than that in Weeks 2 and 3 (P .001 and P .001, respectively). Measurement of cytoplasmic progesterone receptor confirmed that the estrogenic stimulus being applied to the endometrium was potent.^ieng

[Determination of plasma and urinary estrogens by use of a new enzyme method]. / Dosage des oestrogènes plasmatiques et urinaires par une nouvelle méthode enzymatique.

The transhydrogenase function of the oestradiol dehydrogenase of human placenta is used in such a condition as the transhydrogenase activity is directly related to oestrone and oestradiol concentration. The method is highly specific. Reacting oestrone with hydrazin before the assay allows specific determination of oestradiol. The limit of sensitivity is 10 picograms (precision 10%) in plasma and 1 microgram in urine (precision 10%). The determination is done directly on hydrolyzed urine without extraction and is rapid and highly specific. The method seems to be competitive for oestrogen radioimmuno assay in plasma and offers great advantages towards conventional chemical methods in urine. Repetitive profiles of urinary oestrogen excretion can be obtained easely.

Properties of the multiple forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver.

The six forms of the 17alpha-hydroxy steroid dehydrogenase purified from rabbit liver cytosol have very similar physical properties. The molecular weights of all the enzymes were within 3% of the average mol.wt of 39 600. Only one of the six enzymes showed a significant difference in amino acid composition. All but one form of the 17alpha-hydroxy steroid dehydrogenases exhibited greater activities towards the androgen, epitestosterone, than towards oestrogen substrates. With oestrogen substrates one enzyme displayed a high specificity towards the substrate oestradiol-17alpha 3-glucuronide. This high activity was lost if the glucuronic acid moiety was removed or replaced by glucose or galacturonic acid. The other enzyme forms had approximately equal activity toward oestradiol-17alpha and its glucuronide or glucoside derivative. However, substitution of galacturonic acid at C-3 of oestradiol-17alpha substantially decreased the activity of all but one enzyme form.

Metabolism of testosterone 4-14C in skin of castrated rats.

Testosterone metabolism was studied in the skin and preputial glands of normal and prepuberally castrated male rats during the 2nd hair cycle. In catagen-telogen the 17-beta-OHSDH of dorsal skin was higher in castrated than in control animals; 5-alpha-reductase instead, remained unchanged through out the hair cycle. Also in the preputial glands of castrated rats 17-beta-OHSDH was higher than in normal rats. So was 5-alpha-reductase. A possible direct control of a substrate, like Testosterone, and/or of a hypophyseal tropin, like prolactin, on enzymes that direct the metabolism of steroids in target tissues is conceivable.

Synthesis of 16alpha-bromoacetoxyestradiol 3-methyl ether and study of the steroid binding site of human placental estradiol 17beta-dehydrogenase.

Homogeneous estradiol 17beta-dehydrogenase (EC 1.1.1.62) was prepared from human placenta by affinity chromatography and the steroid binding site was studied with affinity-labeling techniques. 16alpha-Bromoacetoxyestradiol 3-methyl ether and the tritated compound were synthesized by condensation of estriol 3-methyl ether with bromoacetic acid or [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide. 16alpha-Bromoacetoxyestradiol 3-methyl ether is stable in 0.01 M phosphate buffer at pH 7.0, 25 degrees, for at least 24 hours. It alkylates cysteine, histidine, methionine, lysine, and tryptophan under physiological conditions. The steroid is a substrate of estradiol 17beta-dehydrogenase, thus it must bind at the steroid binding site. The inactivation of estradiol 17beta-dehydrogenase by 150-fold molar concentrations of 16alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo-first order kinetics with a half-time of 1.5 hours. Estradiol-17beta, NADH, and NADPH slow the rate of inactivation. 2-Mercaptoethanol in molar concentrations 50-fold that of 16alpha-bromoacetoxyestradiol 3-methyl ether stops the inactivation, but does not reverse it. 16alpha-Bromoacetoxyestradiol 3-methyl ether alkylates both NADH and NADPH; the presence of small amounts of enzyme markedly increases the rate of this alkylation. When the enzyme is inactivated with 16alpha-[2-3H]bromoacetoxyestradiol 3-methyl ether, amino acid analysis of acid hydrolysates reveals 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine. Comparison of 28 and 51% inactivated samples indicates that, as inactivation proceeds, the total amount of 3-carboxymethylhistidine decreases, while 1,3-dicarboxymethylhistidine increases, suggesting that the former is converted to the latter by a second alkylation step. When the enzyme is inactivated in the presence of a large excess of NADPH, only 1,3-dicarboxymethylhistidine is found. From the present study it is concluded that estradiol 17beta-dehydrogenase has a histidyl residue present in the catalytic region of the active site as does the previously studied 20beta-hydroxysteroid dehydrogenase.