The relationship between aromatase activity and body fat distribution

BEXWBEN AROMATA!E

~~~

ACi’MTY

AND 3ODY FAT

Dodd w. Icalgcr,*, Eay Pcrel,* Doim Daniilm,* Lii Kiltdip,+ and Wii R.N.Lindseyt ;~m~ct~olSurguy'IbeUniv~otTaodo,McdiulSciaceaBuildloe,

ABSTRACT The metabolism of androstenedione (A)to estrone (El) and 5a-reduced androgens was studied in stromal cells derived from human adipose tissue from different body sites. The tissue was obtained from non-obese patients undergoing cosmetic liposuction or at the time of surgery for rtduction mammoplasty. The conversion of A to E per lx 10 cells was between 6- and 30sfold greater in tRe upper thigh, buttock, and flank than in the abdomen. These differences were present in primary culture and persisted to at least the third subculture. Estrogen formation in breast adipose tissue was similar to that found in cells from abdominal fat. The formation of 5a-reduced metabolites (5 u -androstenedione, androsterone, and dihydrotestosterone) varied from patient to patient but was similar in cells from different body sites. These studies show that the regional distribution of fat may influence the metabolism of androgens in adipose tissue, with upper body fat tending to form a lower ratio of estrogens to 5a -reduced androgens than lower body fat. INTRORUCTION The extraglandular conversion of androgens to estrogens has been shown to be the major source of estrogens in males and post-menopausal females (1-3). This conversion takes place in skin (4), hair (5), muscle (6,7), brain (8), fibroblasts (9-U), adipose tissue (12-l&?),and other sites, but adipose tissue appears to play the central role in peripheral estrogen formation (3).

There is evidence that the stromaf cells of adipose tissue are the major site of aromatase activity and, when grown in culture, the formation of estrogens in these cells can be modulated by a variety of physiological stimuli (16-20). In previous studies, we have shown that breast adipose stromal cells have the capacity to metabolize androstenedione (A) to estrone (El) and a series of Sa-reduced androgens, including Sa-androstanedione (Sa-A-dione), dihydrotestosterone (DHT),

STEROIDS 50 /’ l-3 1987

62

Killinger et al.

and androsterone

(AND).

Estrogen

was dramatically

stimulated

of 10-6 M, while

the formation

either

or modestly

were

unchanged

shown to inhibit

and cortisol

The present the formation adipose

in these cells

at a concentration

of 5Cr-reduced metabolites

increased. formation

conditions

study was designed

of estrogens

stromal

by cortisol

estrogen

stimulated

formation

under both basal

(21). to determine

from androgen

cells was dependent

was

5ff-Reduced androgens

whether

precursors

in

on the site of origin

of these cells. MATERIALS

AND METHODS

Materials Reference steroids obtained from Makor Chemicals Ltd. (Jerusalem, Israel) and the Sigma Chemical Co. (St. Louis, MO) were recrystallized before use. Radioactive steroids h,2,6,7-3H]androstenedione (A) (SA, 85 Ci/mmol), [4-14C]testosterone (T) (SA, 50 mCi/mmol), [4-14C]estrone El (DHT) (SA, 50 mCi/mmol), and [4-14C] dihydrotestosterone (SA, 50 mCi/mmol) were obtained from New England Nuclear Corp. (Boston, MA) and were purified by paper chromatography in the systems hexane-benzene-methanol-water (67:33:80:20) for El and hexane-methanol-water (10:9:1) for T,A, and The 5a-r4-14c]androstane-3, DHT. 17-dione (5c%!-A-dione)and 14C androstepone (AND) were not.commercially available and were prepared in our laboratory as previously described (22). Precoated thin-layer chromatography (TLC) plates, SIL-G UV 254, were purchased from Brinkmann Instruments (Westbury, NY). Whatman no. 1 paper for chromatography was purchased from Canlab (Toronto, Ont). Collagenase (C-2139) was purchased from Sigma Chemical Co. (St. Louis, MO),and trypsin was purchased from Difco Laboratories (Detroit, MI). Patients Studies were carried out on 4 female and 2 male patients. Adipose tissue was obained by liposuction from the abdomen, flank, buttock, and two areas of the upper thigh from a 34year-old female (patient l), from the abdomen, flank, buttock and upper thigh of a 40-year-old female (patient 21, from the abdomen and flank of a 49-year-old female (patient 31, from the abdomen and lower and upper thigh of a 34-year-old female (patient 4), and from the abdomen and flank of two male patients aged 37 and 40 (patients 5 and 6). None of the patients was grossly obese and none had undergone recent weight reduction. Preparation of cells Adipose tissue obtained during liposuction was collected into a 50-mL specimen bottle inserted into the suction line. The tissue was washed with Hank's balanced salt solution

STEROIDS

50 / l-3

1987

AROMATASE

AND BODY FAT DISTRIBUTION

(HBsS), and incubated at 3i"C with 1 mg/mL collagenase in HBSS in a trypsinizing flask (Bellco Glass Inc, Vineland, NJ) for 30 min with constant stirring. The ratio of tissue to collagenase solution was 1:5. After digestion, the tissue was allowed to stand for 10 min and separated into three fractions. The upper layer consisted of mature adipocytes, the middle layer of cells liberated from the stromalvascular fraction, and the lower layer of undigested material. The middle layer was removed with a syringe and centrifuged at 800 x g for 10 min. The pellet was resuspended in 20 mL a-minimal essential medium, supplemented with 15% fetal calf serum and 50~ g/mL amikacin, and transferred to 75 cm2 flasks. Adipose tissue obtained at surgery was minced with scissors prior to treatment with collagenase. The remainder of the cell preparation was identical to that described for liposuction. After 24 h, the attached cells were washed three times with medium and fresh medium was added. The medium was changed every 3-4 days, and after 10 days the cells were confluent and ready for subculture. At the time of subculture, the cells were trypsinized with 10 mL of 0.5% trypsin in citrate saline. The liberated cells were centrifuged, resuspended in medium and transferred to 75-cm2 flasks. The studies described in this report were carried out during the third subculture and the results were obtained from duplicate or triplicate incubations. Cell counts carried out after 24 h in the third subculture revealed a plating efficiency between 40 and 60%. Metabolism studies Studies of A metabolism were carried out between davs 4 and 12 in culture. On the day of the study, the medium was replaced with 10 mL of fresh medium containing PHI-A (20 x lo6 dpm, 100 uM). After 8 h, the steroid metabolites were extracted from-the medium with ethyl acetate (3 vol x 3). [14c]-E, [14c]-T, [14C]-Sa-A-dione, [14cJ-DHT, and [14C]-AND were added to the medium to correct for lasses, and 100 g of each steroid was added to facilitate identification during chromatography. After extraction, phenolic partition was used to separate the metabolites into neutral and phenolic fractions (15). The phenolic fraction was chromatographed on TLC, and the El isolated was further purified by TLC (22). The metabolites in the neutral fracture were separated by TLC and paper chromatography as previously described (22) to yield T, 5CY-A-dione,and a fraction containing both AND and DHT. The radioactivity isolated in each fraction from flasks containing no cells was subtracted before calculating conversion to each product. Recovery from El ranged from 20 to 30%, T from 35 to 47%, 5a-A-dione-from 25*to 35%, and AND and DHT from 20 to 30%. The cells were harvested by trypsinization and counted in a hemocytometer.

STEROIDS

50 / l-3 1987

63

64

Killinger etal.

In the studies in which cortisol was added, the cells were grown in the presence of 10e6 M cortisol during the third subculture, and the medium was changed every third day. Radioactive Isotopes were counted with a dual-label program in a Philips model PW4700 liquid scintillation analyzer using Permafluor (Packard Instruments, Downers Grove, IL) as the scintillation mixture. Quench correction and correction for spillover of 14C into the 3H channel were carried out using an internal standard, quenched controls, and an internal computer program. Under the conditions used, 3H was counted with an efficiency of 28% and 14~ with an efficiency of 57%.

RESULTS The conversion stromal buttock,

of androstenedione

cells derived

from adipose

and flank of patient

(A) to estrone

tissue

(El) in

from the abdomen,

2 is shown in Figure

1.

tII-dL AWomen Bu”ock Flank

F

d

A8

F

(A)

(8) (F)

ABF

2

3

ABF 5

-w

Figure 1. Comparison of the conversion of A to El in adipose stromal cells from the abdomen, flank, and buttock of a 40-year-old female (patient 2) in primary culture and passages 1,2,3, and 5. (Percent conversion per 1x10-6 cells.) In the primary

culture,

El formation

in the flank that in the abdomen, buttock

than in the abdomen.

in estrogen

formation

it was approximately

was 6-fold

and I-fold greater

There was a progressive

after the first passage,

greater in the decrease

and by passage

20% of that found in the primary

STEROIDS

3

culture.

50 / 1-3 1987

AROMATASE

AND BODY FAT DISTRIBUTION

65

The relationship between El formation in cells from abdominal fat and cells from the flank and buttock remained relatively constant from the primary culture to the third subculture.

-

-

Figure 2. Cell growth and A metabolism in stromal cells derived from adipose tissue from the abdomen, lower thigh, and upper thigh of a 34-year-old female (patient 4). Studies were carried out on days 4,7, and 10 in culture. The conversion of A to E (Figure 2A) and to W-reduced metabolites (5xY-A-diode+ AND + DYT) (Figure 2B) was determined after an S-h incubation with H -A. Figure 2 shows the growth of cells derived from adipose tissue from the abdomen, lower thigh, and upper thigh of patient 4.

Studies of El formation and the formation of 5cr-reducedmetabolites of A (5cr-A-dione+ DHT + AND) were

carried out on days 4,7, and 10 in passage 3. The cells grew at a similar rate during culture. Estrone formation was more than 30-fold greater in the upper thigh than in the abdomen at each stage, while cells from the lower thigh had

STEROIDS

50 / l-3 1987

66

Killinger et al.

intermediate androgens

aromatase

was similar

Adipose liposuction

tissue

cells

and estrogen

by

sites were

formation

of 5Wreduced

from different

out in passage

in cells

On day 7 in culture,

in

of El in

and flank was comparable,

in these cells was more from abdominal

metabolites

3 and

from all sites

the formation

buttock,

than in cells derived

formation cells

were carried

from the upper thigh,

greater

Four of these

1.

rate was comparable

(Figure 3).

of 5cY-reduced

area, and the fifth site was the abdomen.

of A metabolism

the growth

The formation

from each site.

from 5 body sites was obtained

from patient

the thigh-buttock Studies

activity. in cells

than 30-fold

fat.

The

of A did not differ

in

sites.

Figure 3. Cell growth and the percent conversion of A to El in stromal cells derived from adipose tissue from the abdomen, flank, buttock, and two sites from the upper thigh of a 34-year-old female (patient 1). Studies were carried out on day J in Sulture. El formation was determined after an 8-h incubation with [ HI-A.

STEROIDS

50 / l-3 1987

AROMATASE AND BODY FAT DISTRIBUTION

COMPARISON OF "A" METABOLISM IN BREAST ADIPOSE TABLE 1. STROMAL CE&LS FROM 4 PATIENTS (piCONVERSION PER 1 x 10 CELLS)

E1

T

SC+A-dione

AND+DHT

BTl-3

0.06

0.22

4.87

0.35

BT2-3

ND

0.42

9.46

0.74

BT3-3

0.03

0.35

4.65

0.53

BT4-3

0.02

0.12

2.16

0.29

aStudies were carried out on day 6 in culture (passage 3). Table 1 shows similar studies on breast adipose tissue stromal cells derived from patients undergoing reduction mammoplasty in passage 3 in culture.

Estrogen

formation under basal conditions was low and was comparable to that found in abdominal adipose stromal cells. The formation of W-reduced androgens was similar to that found in other body sites. Figure 4 compares the conversion of A to El in stromal cells derived from abdominal adipose tissue and cells derived from other sites from 4 female and 2 male patients.

The studies were carried out in passage 3 in culture in the late exponential phase of growth.

The degree of El formation at each site varied

from patient to patient and in each case El formation in cells from the flank-thigh-buttock area was 6- to 30-fold greater than in cells from the abdomen. This pattern was seen in cells from both male and female patients. Figure 5 shows two extremes of fat distribution in the female. The figure on the left shows fat accumulation in the thigh-buttock area characteristic of lower body

STEROIDS

50 / l-3

1987

67

68

Killinger et al.

n

Figure 4. Comparison of the formation of ~~lT~"~e~iSe"ds~~~~l adipose tissue from different body sites of 4 females and 2 males. Abdomen (A) Thigh Buttock 1:; Flank (E)

Figure 5. The effect of fat distribution on androgen metabolism in the female. A preponderance of lower body fat is shown on the left and a preponderance of upper body fat on the right. The numbers between the figures are the percent conversion of A to E found in primary culture in patient 2 (Figure 1) per 10 6 Adipose stromal cells. The figure demonstrates that the location of the adipose tissue may affect the amount of E 1 formed from A in patients with similar body weights.

STEROIDS

50 / l-3

1987

AROMATASE

AND BODY FAT DISTRIBUTION

fat and the figure on the right shows the accumulation of upper body or abdominal fat with decreased fat in the buttocks and thighs commonly seen in males.

The

percent conversion of A to El per lo6 cells from the abdomen, buttock, and flank noted in primary culture in Figure 1 is shown between the diagrams.

The distribution

of fat in 2 females of the same weight could influence peripheral androgen metabolism.

The formation of El

would be less in the patient with upper body fat, while the formation of 5o!-reducedandrogens would be similar. DISCUSSION These studies suggest that the metabolism of A to El in adipose tissue stromal cells is dependent on their site of origin.

The formation of El was higher in stromal

cells from the thigh-buttock area than in cells from abdominal fat. This relationship remained consistent, in spite of wide variations in the aromatization of A from patient to patient. These differences in El formation persisted during subculture, suggesting that they were not related to external factors but were inherent in the cells. The formation of 5(X-reduced metabolites of A did not show any consistent variation relative to the origin of the adipose tissue. Obesity is commonly associated with a decrease in plasma sex hormone-binding globulin (SHBG) and an increase in the percentage of free testosterone (23-24).

It has

also been demonstrated (25) that the location of body fat is important, since there was an inverse relationship between waist:hip ratio and plasma SHBG and a direct relationship between this ratio and free T.

These observations are in keeping with the results of the present study. Adipose stromal cells from all sites were active in the formation of 5cy-reducedmetabolites from A. Cells derived from abdominal fat, however, had a

STEROIDS

50 / l-3 1987

69

70

Killingeret al.

lower

ratio

of El to 5cr-reduced androgens

the thigh-buttock

area,

favor less estrogen SHBG synthesis

androgenism

formation,

which may in turn affect

and co-workers

(27) observed and obesity

presentations.

(26) and McKenna

that patients

One group of patients

a second

group had amenorrhea without

hirsutism

of El and normal

SHBG.

of this study it is possible distribution

of adipose

the clinical

presentation.

without

were

and

with hyper-

may have different

patients levels

from

ratio would

in the liver.

Hosseinian co-workers

than cells

so a high waist:hip

clinical

were hirsute hirsutism.

and The

found to have elevated From the observations

that the regional

tissue may be a factor

in

ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Founcil of Canada, the National Cancer Institute We would also like of Canada, and the Whitney Foundation. to thank Miss Cecilia Millar for her assistance with the manuscript.

NOTE *Please send reprint requests to: Dr. D.W. Killinger, Medical Sciences Building, Room 7366, University of Toronto, Toronto, Canada M5S lA8.

REFERENCES 1.

2.

3.

MacDonald PC, Rombaut P, and Siiteri PK (1967). Plasma precursors of estrogen. 1. Extent of conversion of plasma 4-androstenedione to estrone in normal males and non-pregnant, normal, castrate and adrenalectomized females. J CLIN ENDOCRINOL METAB -27:11031111. Grodin JM, Siiteri PK, and MacDonald PC (1973). Source of estrogen production in postmenopausal women. J CLIN ENDOCRINOL METAB 36:207-214. MacDonald PC, Edman, CD,?emsell DL, Porter YC, and Siiteri PK (1978). Effect of obesity on conversion of plasma androstenedione to estrone in post menopausal women with and without endometrial cancer. AM J OBSTET GYNECOL 130:448-455.

STEROIDS

50 / l-3

1987

AROMATASE

4.

5. 6.

7.

8.

9. 10.

11.

12. 13. 14. 15. 16.

17. 18.

AND BODY FAT DISTRIBUTION

Gomez E, and Hsia S (1968). In vitro metabolism of testosterone and androstenedione in human skin. BIOCHEMISTRY 7:24-32. Schweikert HUT Milewich L, and Wilson JD (1975). Aromatization of androstenedione by isolated human hairs. J CLIN ENDOCRINOL METAB 40:413-417. Longcope C, Pratt JH, SchneidexTH, and Fineberg SE (1976). The in vitro metabolism of androgens by muscle and adipose tissue of normal man. STEROIDS 28:521-533. Gngcope C, Pratt JH, Schneider Sii,and Fineberg SE (1978). Aromatization of androgens by muscle and adinose tissue in vivo. J CLIN ENDOCRINOL METAB 46:i46-152. zftolin F, Ryan DJ, Davis IJ, Reddy W, Flores F, Petro Z, and Kuhn M (1975). The formation of estrosens by central neuroendocrine tissues. RES PROJ HORM RES 31:295-315. Schwzkert I-HI, Milewich L, and Wilson JD (1976). Aromatization of androstenedione by cultured human fibroblasts. J CLIN ENDOCRINOL METAB 43:785-795. Schweikert HU (1979). Conversion of ai&ostenedione to estrone in human fibroblasts cultured from prostate, genital and nongenital skin. HORM METAB RES 11:635-640. BerkGitz GD, Fujimata M, Brown TR, Brodie AM, and Migeon CJ (1984). Aromatase activity in cultured human genital skin fibroblasts. J CLIN ENDOCRINOL METAB 59:665-671. Bolt HAI‘; and Gobel P (1972). Formation of estrosens from androgens by human subcutaneous adipose tissue in vitro. HORM METAB RES 43312-313. Schindler AE, Ebert A, an3 Friedrich E (1975). Conversion of androstenedione to estrone by human fat tissue. J CLIN ENDOCRINOL METAB 35:627-630. Nimrod A, and Ryan K (19z). Aromatization of androgens by human abdominal and breast fat tissue. J CLIN ENDOCRINOL METAB 40:367-372. Perel E, and KillGqer DW (1979). The interconversion and aromatization of androgens by human adipose tissue. Y STEROID BIOCHEM 10:623-627. Cleland WH, Mendelson CR, and Simpson ER (1983). Aromatase activity of membrane fractions of human adipose tissue stromal cells and adipocytes. ENDOCRINOLOGY =:2155-2160. Folkert EJ, and James VHT (1983). Aromatization of steroids in peripheral tissues. J STEROID BIOCHEM 19:687-690. Empson ER, Ackerman GE, Smith ME, and Mendelson CR (1981). Estrogen formation in stromal cells of adipose tissue of women:Induction by glucocorticoids. PROC NATL ACAD SC1 (USA) 2:5690-5694.

STEROIDS

50 I 1-3 1987

71

72

Killinger et a/.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Mendelson CR, Smith ME, Cleland WH, and Simpson ER (1984). Regulation of aromatase activity of cultured adipose stromal cells by catecholamines and adrenocorticotrophin. MOL CEL ENDOCRINOL 37:61-72. Folkert EJ, and Jam= WHT (1984). The action of dexamethasone and prolactin on aromatase activity in human adipose tissue. J STEROID BIOCHEM 20:679-681. Perel E, Daniilescu D, Kindler S, Xfiarlip L, and Killinger DW (1986). The formation of 5cz-reduced androgens in stromal cells from human breast adipose tissue. J CLIN ENDOCRINOL METAB %:314-318, Perel E, and Killinqer DW (1983). The metabolism of androstenedione and testosterone to C metabolites in normal breast, breast carcinoma an a9 benign prostatic hypertrophy tissue. J STEROID BIOCHEM 19:1135-1139. Eass AT, Burman KD, Dohms WT, and Boehm TM (1981). Endocrine function in human obesity. METABOLISM 30:89-104. xymate SR, Fariss BL, Basset ML, and Matej L (1981). Obesity and its role in polycystic ovarian syndrome. J CLIN ENDOCRINOL METAB 52:1246-1248. Evans DJ, Hoffmann RG, Kxkhoff RK, and Kissebah AH (1983). Relationship of androgenic activity to body fat topography, fat cell morphology, and metabolic aberrations in premenopausal women. J CLIN ENDOCRINOL METAB 57:304-310. Hosseizan AH, Kim MH, and Rosenfield RL (1976). Obesity and oligomenorrhea are associated with hyperandrogenism independent of hirsutism. J CLIN ENDOCRINOL METAB -42: 765-769. McKenna JT, Loughlin T, Daly L, Smyth PA, Culliton M, and Cunningham SK (1984). Variable clincial and hormonal manifestations of hyperandrogenemia. METABOLISM -33:714-717.

APPENDIX The following trivial names and abbreviations have been used: Androstenedione [A): 4-androstene-3,17-dione Androsterone (AND): 3a-hydroxy-5cz-androstan-17-one SCY-A-dione: 5a-androstane-3,17-dione Dihydrotestosterone (DHT): 17&hvdroxv-5a-androstan-3-one Estrone (E ): 3-hydroxy-1,3,5'ilOi-estratrien-17-one Testosterofte (T): 17&hydroxv-4-androsten-3-one Cortisol: hydrocortisone 21-sodium succinate

STEROIDS

50 f l-3

1987