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Aromatization of steroids in peripheral tissues
_I srrroid Biochem Vol. 19, No. I. pp. 687-690, Printed in Great Britain. All rights reserved
1983
Copyright
AROMATIZATION
OF
PERIPHERAL
STEROIDS
0
0022-473 l/83 $3.00 + 0.00 1983 Pergamon Press Ltd
IN
TISSUES
E. J. FOLKERD and V. H. T. JAMES Department of Chemical Pathology, St Mary’s Hospital Medical School. London W2 IPG, England SUMMARY The action of glucocorticoids on aromatase activity in human adipose tissue has been investigated. Oestrogen production was increased in tissue maintained in the presence of dexamethasone and cortisol. Dexamethasone was effective at concentrations as low as 1 nmol/l. In contrast, over the 40 h incubation period, cortisol was only effective at concentrations in excess of 500 nmol/l, although after conditions of prolonged incubation, induction of aromatase activity was measurable using cortisol concentrations as low as 28 nmol/l. There were apparent variations in both basal levels of aromatase activity and in response to glucocorticoids in adipose tissue taken from various sites within the body.
INTRODUCTION
(5:4:1) were used as solvent systems. All cofactors, Penicillin-G (1970 units/mg) and Streptomycin sulphate were obtained from Sigma (London) Chemical Co. Ltd. Medium 199 with Earle’s salts containing L-glutamine and 2.2 g/l sodium bicarbonate and foetal calf’s serum were obtained from Gibco:Bio-Cult Ltd, Paisley.
The peripheral aromatization of androgens to oestrogens provides a source of circulating oestrogen which is of particular importance in men and postmenopausal women and also provides oestrogens which can act locally within the cell in which they are produced or in surrounding tissue. Abnormalities in aromatase activity would alter the circulating levels of steroids available to target tissues and also change the intracellular balance of androgens and oestrogens. It is therefore important to measure the normal activity of the enzyme in peripheral tissues and to determine the mechanism of control of enzyme activity. In gonadal tissue aromatase activity can be induced by the action of gonadotrophins [l, 23. However, in adipose tissue, one of the major sites of peripheral aromatization, we have been unable to demonstrate that such a mechanism is operative [3]. Recently, it has been reported [4] that the synthetic glucocorticoid, dexamethasone, will increase aromatase activity in the stromal-vascular cells of adipose tissue while maintained in tissue culture. In the present study we have examined the action of glucocorticoids on explants of whole adipose tissue taken at surgery and kept in culture for short periods of time.
Measurement
ofaromatase
activity
The conversion of androstenedione to oestrone was measured in adipose tissue slices or homogenates using a technique described in detail elsewhere [S]. In brief, [7n-3H]-androstenedione (100 pmol, 1 &i) and [4-14C]-oestrone (400 pmol, 0.02 &i) were incubated with tissue in 0.1 M phosphate buffer pH 7.4 in a total volume of 1 ml, for 3 h at 37°C in the presence of cofactors for the generation of NADPH. Product oestrone was extracted and purified by paper and thinlayer chromatography. When tissue slices were used it was necessary to remove lipid from the ether extracts of the tissue. This was achieved by adding methanol:water (75:25) to the dried extracts and cooling the mixture until the lipid solidified. After removal of the methanol: water extract, the process was repeated twice more. The remaining lipid was discarded and the methanol fractions pooled and extracted with an equal volume of ether, 3 times. The ether extracts were dried before purification of the oestrone by chromatography.
EXPERIMENTAL Chemicals [7n-3H]-androstenedione (15 Ci/nmol) and obtained from [4-i4C]-oestrone (55 mCi/nmol), Radiochemicals International, Amersham, U.K. were purified before use by paper chromatography. Light fraction): toluene : methanol : petroleum (100-l 20” n-heptane: methanol : water water (1:4:4: 1) and Correspondence to: Prof. V. H. T. James, Department of Chemical Pathology, St Mary’s Hospital Medical School, London W2 IPG, England. 687
Short term culture
ofadipose tissue
Fresh adipose tissue slices (5-10 mg) were cut freehand and transferred to flasks containing 5 ml Medium 199 containing Penicillin-G (100 U/ml), Streptomycin sulphate (100 pg/ml) and foetal calf serum (10%). Approximately 500 mg of tissue was added to each flask and allowed to float. The gas phase was air. The flasks were covered and incubated at 37°C. Variations of these conditions are described
E. J. FOLKERD and V. H. T. JAMES
688
Fig. I, Subcutaneous adipose tissue was maintained in the presence of various concentrations of cortisol or dexamethasone for 40 h at 37”C, before the assay of aromatase activity. Oestrone production is expressed as a percentage of the result obtained when the tissue was maintained in the absence of added glucocorticoid.
in the text. After
a period of incubation the tissue slices were removed and resuspended in 1 ml 0.1 M phosphate buffer prior to measurements of aromatase activity. Used culture medium was also checked for aromatase activity.
by glucocorticoids
As shown in Fig. 1 the aromatase enzyme was induced in whole adipose tissue in uitro by dexamethasone and cortisol. Dexamethasone increased oestrogen production at concentrations as low as 1 nmol/l. In contrast, over the 40 h incubation period cortisol was only effective at concentrations in excess of 500 nmol/l. Induction of aromatase
Regional variations in response to glucocorticoids In one subject adipose tissue was removed from three sites, subcutaneous from the area of the umbilicus, extra peritoneal and from the groin. As shown in Fig. 2 basal levels of activity and response to dexamethasone and cortisol differed in tissues from different areas.
Induction tissue
RESULTS
Induction ofaromatase
providing that the culture medium was replaced at 45 h with the same medium that was added initially. Depending on the equilibrium constant for the binding of cortisol to its cytoplasmic receptor and the ability of adipose tissue to store glucocorticoids, it is possible that if a substantial amount of the cortisol added initially was taken up by the tissue by 45 h, then replacement of the medium may have effectively increased the overall cortisol concentration, perhaps raising it to one which would induce the enzyme over the remaining time available. Oestrogen activity was not increased if the medium was replaced with cortisol free medium at 45 h. Failure to replace the medium resulted in loss of enzyme activity and possibly tissue death.
by low concentrations
of aromatase
Table
of corti-
I, The effect of low cortisol concentration
No. I 2 3 4 5 6 7
0 14 28 42 0 42 84 Used culture
Cortisol concentration (nmol/l)
(Medium (Medium (Medium medium
unchanged) unchanged) unchanged)
in omental
adipose
Simpson et al.[4] were unable to demonstrate any effect of dexamethasone on stromal cells from human omental adipose tissue. In contrast, in the present study, using whole adipose tissue, the action of dexamethasone was quite marked, as was that of cortisol (Fig. 3) although in general the magnitude of response was lower in omental fat compared with adipose tissue taken from other sites. DISCUSSION
SO1
Induction of aromatase in adipose tissue was possible using cortisol concentrations in the normal range for unbound plasma cortisol (i.e. 7-70nmol/l) after prolonged periods of incubation as shown in Table 1,
activity
The extent of peripheral conversion of androstenedione to oestrone will depend on the availability of reduced NADP, androstenedione and aromatase. The redox state of the cofactor in stromal cells of adipose
on oestrogen
production
in adipose
tissue
Aromatase activity at 45 h (pg/O.5 g tissue/3 h)
Aromatase activity at llOh (pg/O.5 g tissue/3 h)
40.6 62.3 43.3 37.2 40.5 40.5 40.5 0.3*
225.3 119.4 414.1 680.1 3.9 3.2 18.4 0.3
* (pg/ml/3 h). The induction of aromatase activity in abdominal adipose tissue in the presence of low cortisol concentrations. Duplicate samples of adipose tissue (0.5g) were maintained in tissue culture in the presence or absence of cortisol as shown. Aromatase activity was measured at 45 h, and 110 h in the duplicate sample. In samples l-4 the tissue culture medium was replaced at 45 h with the same medium added initially. In samples 5-7 the medium was not replaced after 45 h.
689
Aromatization in peripheral tissues
Fig. 2. Adipose tissue was taken from three different sites, subcutaneous from the umbilicus, extraperitoneal and from the groin of a male subject undergoing surgery for hernia repair. Tissue was maintained in the presence or absence of cortisol or dexamethasone for 40 h at 37°C before assay of aromatase activity as described in the methods.
tissue is not known as far as we are aware and merits further study. The availability of substrate is probably one of the most important factors influencing the control of aromatase. Serum concentrations of androstenedione in both men and women are usually between 1 and 10 nmol/l and as such are rather lower than the apparent Michaelis Constant for androstenedione (0.025-2.0 PM) as measured in a number of tissues [&lo]. Even taking into account measurements of tissue levels of steroids which indicate that androstenedione concentrations in adipose tissue may be 2-3 times greater than in plasma [ll], substrate concentrations will still be a limiting factor for the reaction. Production of androstenedione by the adrenals and the gonads, clearance by the liver, binding by plasma proteins and alternative pathways of metabolism will all influence the amount of androstenedione available for aromatization. Likely routes of depletion are conversion to testosterone or reduction to See-reduced metabolites. In adipose tissue, in vitro, androstenedione is readily converted to testosterone (unpublished observations), although we have been unable to demonstrate any aromatization of testosterone to oestradiol. Little is known about the Sa-reductase enzyme in human adipose tissue but in placenta See-reduced metabolites have been shown to inhibit aromatase activity [12] and therefore substances inducing the synthesis of Sa-reductase may diminish aromatization whereas substances inhibiting Sa-reductase will promote aromatization providing an adequate supply of NADPH is available. Factors which act to increase enzyme synthesis will lead to increased oestrogen production if other factors are not limiting. As shown by Simpson et aI.[4] and confirmed in the present study, glucocorticoids appear to induce aromatase activity in adipose tissue in vitro. Whether the reaction will take place at physiological concentrations of cortisol is still unclear. It is difficult to equate results obtained in
vitro with the situation in uiuo where the tissue is continuously perfused with plasma containing cortisol and many other substances which might modulate its action. If glucocorticoids are able to induce aromatase in adipose tissue in uiuo then one would assume that conditions in which adrenal output of cortisol is raised might be associated with evidence of increased oestrogen production. However, Hsueh and Erikson[13] have reported that concentration of dexamethasone and cortisol of the same order as those found to induce aromatase activity in adipose tissue in vitro, inhibit induction of FSH in the granulosa cells of the rat ovary. If the same is true in human gonadal tissue, then in times of glucocorticoid excess in premenopausal women, ovarian oestrogen production would be inhibited while peripheral oestrogen production would be stimulated. Therefore, although the overall hormonal profile may not be one of oestrogen excess, within specific tissues alterations in hormone production might be measurable. As reported previously [14] we have found a moderately higher level of aromatase activity in abdominal fat from 2 women with Cushing’s syndrome compared with that found in abdominal fat from normal women. In contrast to the findings of Simpson et a/.[43 we were unable to demonstrate an increase in oestrone production by omental tissue maintained in medium with added glucocorticoid although the response was reduced compared to that obtained using subcutaneous adipose tissue. The reason for this discrepancy is not clear, although the difference in tissue culture conditions in the two studies is a likely cause. Regional variations in base1 levels of aromatase activity and in response to glucocorticoids in adipose tissue were apparent. Whether these differences are a true reflection of enzyme activities within the tissues in uiuo or merely differences in response to the conditions of culture are unknown. It has been suggested [15] that adipose tissue from the omentum is more
active
in metabolizing
cortisol
than
subcuta-
Fig. 3. Omental adipose tissue was obtained at surgery from a female subject. The tissue was maintained in the presence or absence of cortisol or dexamethasone for 40 h at 37°C before the assay of aromatase activity.
E.J.
690
FOLKERD and V.H.T.
neous adipose tisssue and therefore it seems likely that in omental tissue cortisol might be metabolized before induction of aromatase can take place. If such regional variations in activity do exist in uiuo, it would seem likely that adipose tissue might have a significant role to play in local oestrogen production rather than mainly contributing to the circulating pool of oestrogens. Acknowlrdyements~This work was supported by a grant from the Campaign for Cancer Research. We gratefully acknowledge the assistance of Dr P. A. Beranek and staff at the St Charles Hospital, London in supplying the samples of adipose tissue used in this investigation. REFERENCES 1. Moon Y. S., Tsang B. K., Simpson C. and Armstrong D. T.: 17/&Estradiol biosynthesis in cultured granulosa and thecal cells of human ovarian follicles: stimulation by follicle stimulating hormone. J. c/in. Endocr. Metab. 47 (1978) 263-267. 2. Erickson G. F., Hsueh A. J. W., Quigley M. E., Rebar R. W. and Yen S. S. C.: Functional studies of aromatase activity in human granulosa cells from normal and polycystic ovaries. J. c/in. Endow. Metab. 49 (1979) 514-519. 3. Folkerd E. J., Jacobs H. S., Van der Spuy 2. and James V. H. T.: Failure of FSH to influence aromatization in human adipose tissue. Clin. Endocr. 16 (1982) 621-625. 4. Simpson E. R., Ackerman G. E.. Smith M. E. and Mendelson C. R.: Estrogen formation in stromal cells of adipose tissue of women: induction by glucocorticosteroids. Proc. mtn. Acad. Sci. US-Biol Sci. 78 (1981) 569c-5694. 5. Folkerd E. J., Reed M. J. and James V. H. T.: Oestrogen production in adipose tissue from normal women
JAMES
and women with endometrial cancer in vitro. J. steroid Biochem. 16 (1982) 297-302. 6. Forney J. P., Milewich L., Chen G. T., Garlock J. L., Schwartz B. E., Edman C. D. and MacDonald P. C.: Aromatization of androstenedione to oestrone by human adipose tissue in vitro. Correlation with adipose tissue mass, age and endometrial neoplasia. J. clin. Endocr. Metab. 53 (1981) 192-199. I. Ackerman G. E., Smith M. E., Mendelson C. R., MacDonald P. C. and Simpson E. R.: Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J. c/in. Endocr. Metab. 53 (1981) 412-417. 8. Thompson E. A. and Siiteri P. K.: Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J. biol. Chem. 249 (1974) 5364-5372. 10. Schweikert H. U.. Milewich L. and Wilson J. D.: Aromatization of androstenedione by cultured human fibroblasts. J. clin. Endocr. Metab. 43 (1976) 785 795. II. Feher T.: Role of adipose tissue in the production and metabolism of androgens. Proceedings of the 9th Congress of‘ the Hungarian Society of Endocrinology. Szeged, Hungary (1979) pp. 251-257. 12. Siiteri P. K. and Thompson E. A.: Studies of human placental aromatase. J. steroid Biochem. 6 (1975) 317-322. 13. Hsueh A. J. W. and Erikson G. F.: Glucocorticoid inhibition of FSH-induced estrogen production in cultured rat granulosa cells. Steroids 32 (1978) 639-647. 14. James V. H. T., Folkerd E. J., Bonney R. C., Beranek P. M. and Reed M. J.: Factors influencing oestrogen production and metabolism in postmenopausal women with endocrine cancer. J. endocr. Invest. 5 (1982) 335-345. 15. Karl H. J., Raith L. and Engelhardt E.: In vitro transformation of cortisol by human adipose tissue. Harm. Metab. Res. I (1969) 95.
1983
Copyright
AROMATIZATION
OF
PERIPHERAL
STEROIDS
0
0022-473 l/83 $3.00 + 0.00 1983 Pergamon Press Ltd
IN
TISSUES
E. J. FOLKERD and V. H. T. JAMES Department of Chemical Pathology, St Mary’s Hospital Medical School. London W2 IPG, England SUMMARY The action of glucocorticoids on aromatase activity in human adipose tissue has been investigated. Oestrogen production was increased in tissue maintained in the presence of dexamethasone and cortisol. Dexamethasone was effective at concentrations as low as 1 nmol/l. In contrast, over the 40 h incubation period, cortisol was only effective at concentrations in excess of 500 nmol/l, although after conditions of prolonged incubation, induction of aromatase activity was measurable using cortisol concentrations as low as 28 nmol/l. There were apparent variations in both basal levels of aromatase activity and in response to glucocorticoids in adipose tissue taken from various sites within the body.
INTRODUCTION
(5:4:1) were used as solvent systems. All cofactors, Penicillin-G (1970 units/mg) and Streptomycin sulphate were obtained from Sigma (London) Chemical Co. Ltd. Medium 199 with Earle’s salts containing L-glutamine and 2.2 g/l sodium bicarbonate and foetal calf’s serum were obtained from Gibco:Bio-Cult Ltd, Paisley.
The peripheral aromatization of androgens to oestrogens provides a source of circulating oestrogen which is of particular importance in men and postmenopausal women and also provides oestrogens which can act locally within the cell in which they are produced or in surrounding tissue. Abnormalities in aromatase activity would alter the circulating levels of steroids available to target tissues and also change the intracellular balance of androgens and oestrogens. It is therefore important to measure the normal activity of the enzyme in peripheral tissues and to determine the mechanism of control of enzyme activity. In gonadal tissue aromatase activity can be induced by the action of gonadotrophins [l, 23. However, in adipose tissue, one of the major sites of peripheral aromatization, we have been unable to demonstrate that such a mechanism is operative [3]. Recently, it has been reported [4] that the synthetic glucocorticoid, dexamethasone, will increase aromatase activity in the stromal-vascular cells of adipose tissue while maintained in tissue culture. In the present study we have examined the action of glucocorticoids on explants of whole adipose tissue taken at surgery and kept in culture for short periods of time.
Measurement
ofaromatase
activity
The conversion of androstenedione to oestrone was measured in adipose tissue slices or homogenates using a technique described in detail elsewhere [S]. In brief, [7n-3H]-androstenedione (100 pmol, 1 &i) and [4-14C]-oestrone (400 pmol, 0.02 &i) were incubated with tissue in 0.1 M phosphate buffer pH 7.4 in a total volume of 1 ml, for 3 h at 37°C in the presence of cofactors for the generation of NADPH. Product oestrone was extracted and purified by paper and thinlayer chromatography. When tissue slices were used it was necessary to remove lipid from the ether extracts of the tissue. This was achieved by adding methanol:water (75:25) to the dried extracts and cooling the mixture until the lipid solidified. After removal of the methanol: water extract, the process was repeated twice more. The remaining lipid was discarded and the methanol fractions pooled and extracted with an equal volume of ether, 3 times. The ether extracts were dried before purification of the oestrone by chromatography.
EXPERIMENTAL Chemicals [7n-3H]-androstenedione (15 Ci/nmol) and obtained from [4-i4C]-oestrone (55 mCi/nmol), Radiochemicals International, Amersham, U.K. were purified before use by paper chromatography. Light fraction): toluene : methanol : petroleum (100-l 20” n-heptane: methanol : water water (1:4:4: 1) and Correspondence to: Prof. V. H. T. James, Department of Chemical Pathology, St Mary’s Hospital Medical School, London W2 IPG, England. 687
Short term culture
ofadipose tissue
Fresh adipose tissue slices (5-10 mg) were cut freehand and transferred to flasks containing 5 ml Medium 199 containing Penicillin-G (100 U/ml), Streptomycin sulphate (100 pg/ml) and foetal calf serum (10%). Approximately 500 mg of tissue was added to each flask and allowed to float. The gas phase was air. The flasks were covered and incubated at 37°C. Variations of these conditions are described
E. J. FOLKERD and V. H. T. JAMES
688
Fig. I, Subcutaneous adipose tissue was maintained in the presence of various concentrations of cortisol or dexamethasone for 40 h at 37”C, before the assay of aromatase activity. Oestrone production is expressed as a percentage of the result obtained when the tissue was maintained in the absence of added glucocorticoid.
in the text. After
a period of incubation the tissue slices were removed and resuspended in 1 ml 0.1 M phosphate buffer prior to measurements of aromatase activity. Used culture medium was also checked for aromatase activity.
by glucocorticoids
As shown in Fig. 1 the aromatase enzyme was induced in whole adipose tissue in uitro by dexamethasone and cortisol. Dexamethasone increased oestrogen production at concentrations as low as 1 nmol/l. In contrast, over the 40 h incubation period cortisol was only effective at concentrations in excess of 500 nmol/l. Induction of aromatase
Regional variations in response to glucocorticoids In one subject adipose tissue was removed from three sites, subcutaneous from the area of the umbilicus, extra peritoneal and from the groin. As shown in Fig. 2 basal levels of activity and response to dexamethasone and cortisol differed in tissues from different areas.
Induction tissue
RESULTS
Induction ofaromatase
providing that the culture medium was replaced at 45 h with the same medium that was added initially. Depending on the equilibrium constant for the binding of cortisol to its cytoplasmic receptor and the ability of adipose tissue to store glucocorticoids, it is possible that if a substantial amount of the cortisol added initially was taken up by the tissue by 45 h, then replacement of the medium may have effectively increased the overall cortisol concentration, perhaps raising it to one which would induce the enzyme over the remaining time available. Oestrogen activity was not increased if the medium was replaced with cortisol free medium at 45 h. Failure to replace the medium resulted in loss of enzyme activity and possibly tissue death.
by low concentrations
of aromatase
Table
of corti-
I, The effect of low cortisol concentration
No. I 2 3 4 5 6 7
0 14 28 42 0 42 84 Used culture
Cortisol concentration (nmol/l)
(Medium (Medium (Medium medium
unchanged) unchanged) unchanged)
in omental
adipose
Simpson et al.[4] were unable to demonstrate any effect of dexamethasone on stromal cells from human omental adipose tissue. In contrast, in the present study, using whole adipose tissue, the action of dexamethasone was quite marked, as was that of cortisol (Fig. 3) although in general the magnitude of response was lower in omental fat compared with adipose tissue taken from other sites. DISCUSSION
SO1
Induction of aromatase in adipose tissue was possible using cortisol concentrations in the normal range for unbound plasma cortisol (i.e. 7-70nmol/l) after prolonged periods of incubation as shown in Table 1,
activity
The extent of peripheral conversion of androstenedione to oestrone will depend on the availability of reduced NADP, androstenedione and aromatase. The redox state of the cofactor in stromal cells of adipose
on oestrogen
production
in adipose
tissue
Aromatase activity at 45 h (pg/O.5 g tissue/3 h)
Aromatase activity at llOh (pg/O.5 g tissue/3 h)
40.6 62.3 43.3 37.2 40.5 40.5 40.5 0.3*
225.3 119.4 414.1 680.1 3.9 3.2 18.4 0.3
* (pg/ml/3 h). The induction of aromatase activity in abdominal adipose tissue in the presence of low cortisol concentrations. Duplicate samples of adipose tissue (0.5g) were maintained in tissue culture in the presence or absence of cortisol as shown. Aromatase activity was measured at 45 h, and 110 h in the duplicate sample. In samples l-4 the tissue culture medium was replaced at 45 h with the same medium added initially. In samples 5-7 the medium was not replaced after 45 h.
689
Aromatization in peripheral tissues
Fig. 2. Adipose tissue was taken from three different sites, subcutaneous from the umbilicus, extraperitoneal and from the groin of a male subject undergoing surgery for hernia repair. Tissue was maintained in the presence or absence of cortisol or dexamethasone for 40 h at 37°C before assay of aromatase activity as described in the methods.
tissue is not known as far as we are aware and merits further study. The availability of substrate is probably one of the most important factors influencing the control of aromatase. Serum concentrations of androstenedione in both men and women are usually between 1 and 10 nmol/l and as such are rather lower than the apparent Michaelis Constant for androstenedione (0.025-2.0 PM) as measured in a number of tissues [&lo]. Even taking into account measurements of tissue levels of steroids which indicate that androstenedione concentrations in adipose tissue may be 2-3 times greater than in plasma [ll], substrate concentrations will still be a limiting factor for the reaction. Production of androstenedione by the adrenals and the gonads, clearance by the liver, binding by plasma proteins and alternative pathways of metabolism will all influence the amount of androstenedione available for aromatization. Likely routes of depletion are conversion to testosterone or reduction to See-reduced metabolites. In adipose tissue, in vitro, androstenedione is readily converted to testosterone (unpublished observations), although we have been unable to demonstrate any aromatization of testosterone to oestradiol. Little is known about the Sa-reductase enzyme in human adipose tissue but in placenta See-reduced metabolites have been shown to inhibit aromatase activity [12] and therefore substances inducing the synthesis of Sa-reductase may diminish aromatization whereas substances inhibiting Sa-reductase will promote aromatization providing an adequate supply of NADPH is available. Factors which act to increase enzyme synthesis will lead to increased oestrogen production if other factors are not limiting. As shown by Simpson et aI.[4] and confirmed in the present study, glucocorticoids appear to induce aromatase activity in adipose tissue in vitro. Whether the reaction will take place at physiological concentrations of cortisol is still unclear. It is difficult to equate results obtained in
vitro with the situation in uiuo where the tissue is continuously perfused with plasma containing cortisol and many other substances which might modulate its action. If glucocorticoids are able to induce aromatase in adipose tissue in uiuo then one would assume that conditions in which adrenal output of cortisol is raised might be associated with evidence of increased oestrogen production. However, Hsueh and Erikson[13] have reported that concentration of dexamethasone and cortisol of the same order as those found to induce aromatase activity in adipose tissue in vitro, inhibit induction of FSH in the granulosa cells of the rat ovary. If the same is true in human gonadal tissue, then in times of glucocorticoid excess in premenopausal women, ovarian oestrogen production would be inhibited while peripheral oestrogen production would be stimulated. Therefore, although the overall hormonal profile may not be one of oestrogen excess, within specific tissues alterations in hormone production might be measurable. As reported previously [14] we have found a moderately higher level of aromatase activity in abdominal fat from 2 women with Cushing’s syndrome compared with that found in abdominal fat from normal women. In contrast to the findings of Simpson et a/.[43 we were unable to demonstrate an increase in oestrone production by omental tissue maintained in medium with added glucocorticoid although the response was reduced compared to that obtained using subcutaneous adipose tissue. The reason for this discrepancy is not clear, although the difference in tissue culture conditions in the two studies is a likely cause. Regional variations in base1 levels of aromatase activity and in response to glucocorticoids in adipose tissue were apparent. Whether these differences are a true reflection of enzyme activities within the tissues in uiuo or merely differences in response to the conditions of culture are unknown. It has been suggested [15] that adipose tissue from the omentum is more
active
in metabolizing
cortisol
than
subcuta-
Fig. 3. Omental adipose tissue was obtained at surgery from a female subject. The tissue was maintained in the presence or absence of cortisol or dexamethasone for 40 h at 37°C before the assay of aromatase activity.
E.J.
690
FOLKERD and V.H.T.
neous adipose tisssue and therefore it seems likely that in omental tissue cortisol might be metabolized before induction of aromatase can take place. If such regional variations in activity do exist in uiuo, it would seem likely that adipose tissue might have a significant role to play in local oestrogen production rather than mainly contributing to the circulating pool of oestrogens. Acknowlrdyements~This work was supported by a grant from the Campaign for Cancer Research. We gratefully acknowledge the assistance of Dr P. A. Beranek and staff at the St Charles Hospital, London in supplying the samples of adipose tissue used in this investigation. REFERENCES 1. Moon Y. S., Tsang B. K., Simpson C. and Armstrong D. T.: 17/&Estradiol biosynthesis in cultured granulosa and thecal cells of human ovarian follicles: stimulation by follicle stimulating hormone. J. c/in. Endocr. Metab. 47 (1978) 263-267. 2. Erickson G. F., Hsueh A. J. W., Quigley M. E., Rebar R. W. and Yen S. S. C.: Functional studies of aromatase activity in human granulosa cells from normal and polycystic ovaries. J. c/in. Endow. Metab. 49 (1979) 514-519. 3. Folkerd E. J., Jacobs H. S., Van der Spuy 2. and James V. H. T.: Failure of FSH to influence aromatization in human adipose tissue. Clin. Endocr. 16 (1982) 621-625. 4. Simpson E. R., Ackerman G. E.. Smith M. E. and Mendelson C. R.: Estrogen formation in stromal cells of adipose tissue of women: induction by glucocorticosteroids. Proc. mtn. Acad. Sci. US-Biol Sci. 78 (1981) 569c-5694. 5. Folkerd E. J., Reed M. J. and James V. H. T.: Oestrogen production in adipose tissue from normal women
JAMES
and women with endometrial cancer in vitro. J. steroid Biochem. 16 (1982) 297-302. 6. Forney J. P., Milewich L., Chen G. T., Garlock J. L., Schwartz B. E., Edman C. D. and MacDonald P. C.: Aromatization of androstenedione to oestrone by human adipose tissue in vitro. Correlation with adipose tissue mass, age and endometrial neoplasia. J. clin. Endocr. Metab. 53 (1981) 192-199. I. Ackerman G. E., Smith M. E., Mendelson C. R., MacDonald P. C. and Simpson E. R.: Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J. c/in. Endocr. Metab. 53 (1981) 412-417. 8. Thompson E. A. and Siiteri P. K.: Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J. biol. Chem. 249 (1974) 5364-5372. 10. Schweikert H. U.. Milewich L. and Wilson J. D.: Aromatization of androstenedione by cultured human fibroblasts. J. clin. Endocr. Metab. 43 (1976) 785 795. II. Feher T.: Role of adipose tissue in the production and metabolism of androgens. Proceedings of the 9th Congress of‘ the Hungarian Society of Endocrinology. Szeged, Hungary (1979) pp. 251-257. 12. Siiteri P. K. and Thompson E. A.: Studies of human placental aromatase. J. steroid Biochem. 6 (1975) 317-322. 13. Hsueh A. J. W. and Erikson G. F.: Glucocorticoid inhibition of FSH-induced estrogen production in cultured rat granulosa cells. Steroids 32 (1978) 639-647. 14. James V. H. T., Folkerd E. J., Bonney R. C., Beranek P. M. and Reed M. J.: Factors influencing oestrogen production and metabolism in postmenopausal women with endocrine cancer. J. endocr. Invest. 5 (1982) 335-345. 15. Karl H. J., Raith L. and Engelhardt E.: In vitro transformation of cortisol by human adipose tissue. Harm. Metab. Res. I (1969) 95.