Molecular and Cellular Endocrinology, 85 (lY92) 13- 19 0 1992 Elsevier
MOLCEL
Scientific
Publishers
Ireland,
13
Ltd. 0303.7207/92/$0.5.00
02736
Effects of in vivo estrogen treatment on adipose tissue metabolism and nuclear estrogen receptor binding in isolated rat adipocytes Steen B. Pedersen, Jens D. Borglum, Torben Moller-Pedersen
and Bjarn Richelsen
l’nicersity Clinic of Endocrinology and Internal Medicine, Aarhus Amtssygehus, Tage Hansensgade, DK-8000 Aarhus C, Denmark (Received
Key words: Estrogen;
Adipose
6 December
tissue metabolism;
Nuclear
1991; accepted
estrogen
receptor
27 December
binding;
1991)
Adipocyte;
(Rat)
Summary We have previously demonstrated the existence of nuclear estrogen receptors in isolated adipocytes (Pedersen et al. (1991) Biochim. Biophys. Acta 1093, 80-86). In the present study we have investigated the regulatory properties of these nuclear estrogen receptors, in addition to the metabolic effects of estrogen on adipose tissue metabolism. Estrogen treatment (20 pg 17/Sestradiol in NaCl for 7 days) decreased lipoprotein lipase activity (LPL) in the adipose tissue by 62% (p < 0.051, decreased adipocyte size by 27% (p < 0.01) and diminished the normal postovariectomy weight gain. Furthermore, estrogen treatment increased the nuclear estrogen receptor binding in adipocytes; in addition, there was a tendency for increased cytosolic estrogen receptor content as well. Time course studies revealed that already 6 h after a single estrogen injection the B,,, increased from 3.82 k 0.3 fmol/106 cells to 9.8 f 3.6 fmol/106 cells (p < 0.1) and 24 h after a single injection the B,,, was maximally increased to 12.7 k 5.5 fmol/lO’ cells (p < 0.05). The K, was similar at all time points (about 3-5 nM). Furthermore, the specific insulin receptor binding was increased in adipocytes from estrogen treated rats. The specific insulin binding was maximally increased by 149 f 6% (p < 0.001) after 4 days of daily estrogen injections. The increased binding seemed to be due to an increased number of insulin receptors on adipocytes from estrogen treated rats with no alteration of the ED,, value. In conclusion it was found that estrogen treatment has a positive feedback effect on its own nuclear receptor. In addition, estrogen treatment was found to have several metabolic effects in the adipose tissue. Estrogen treatment increased the insulin receptor number, decreased LPL activity, increased the lipolytic response in adipocytes and finally decreased body weight and adipocyte cell size.
Introduction The health consequences of obesity have been correlated with the accumulation of adipose tis-
Correspondence to: Steen B. Pedersen, University Clinic of Endocrinology and Internal Medicine, Aarhus Amtssygebus, Tage Hansensgade, DK-8000 Aarhus C, Denmark.
sue in the abdominal region (android type of obesity), especially accumulation of intraabdominal adipose tissue, whereas the accumulation of adipose tissue in the gluteal and femoral regions (gynoid type) has been considered much less hazardous (Bjiirntorp, 1988; Lundgreen et al., 1989). The factors responsible for the distribution of adipose tissue are mostly unknown but the distribution is in some way sexually linked (Krotkiew-
14
ski et al., 1983; Fried and Kral, 1987; Freedman et al., 1990). Furthermore, estrogen treatment has been shown to affect both the fat distribution (Vague et al., 1984) and the metabolism of isolated fat cells (Ackerman et al., 1981; Benoit et al., 1982; Pecquery et al., 1986; Rebuff&Strive, 1987a; Pasquier et al., 1988). Whether the effects of estrogen on the adipose tissue are mediated by direct or indirect mechanisms is presently unknown (Rebuff&&rive et al., 1990). However, we and other groups have found estrogen receptors in isolated adipocytes, either cytoplasmic receptors (Wade and Gray, 1978; Gray and Wade, 1980; Rebuff&Strive, 1987a) or nuclear receptors (Gray et al., 1981; Pedersen et al., 1991). Thus, it may be suggested that estrogen may have direct effects on the adipocytes through these receptors. Studies in other tissues have demonstrated the existence of non-functional steroid receptors (Gehring and Tomkins, 1974; Boyd-Leinen et al., 1982; Spelsberg et al., 1987; Colvard et al., 19881, i.e. cytoplasmic receptors that are able to bind the steroid but unable to become activated and tightly bound to the nucleus. Thus, methods based on receptor assay after lysis of the cell may yield results that do not correlate to biological effects of the steroid in the given cell type (MacFarlane et al., 1980). Recently, methods have become available that quantitate the number of functional receptors (Spelsberg et al., 1987; Colvard et al., 1988). In a recent study we found that the nuclear estrogen binding in isolated adipocytes could be modulated by various hormones such as catecholamines and insulin (Pedersen et al., 1991). In order to get more insight in the factors which regulate the number and the affinity of the nuclear estrogen receptor we investigated the effects of estrogen treatment on these parameters. In addition we investigated the effect of estrogen treatment on the metabolism of the adipose tissue (lipoprotein lipase activity, insulin receptor binding and lipolytic activity). Materials
and methods
Chemicals 17@[2,4,6,7,16,17-“HlEstradiol, 140-170 Ci/mmol, was obtained
spec. act. = from Amersham
(UK). [Carboxy/-‘“ClTriolein, spec. act. = 112 mCi/mmol, was purchased from NEN Research Products and [12sI]insulin was kindly donated by Novo-Nordisk Research Institute. Crude collagenase from Clostridium histeolyticum was obtained from Worthington Biochemical Corp., Freehold, NJ, USA. Estradiol benzoate and sesame oleum were obtained as pharmaceutical. All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Animals Female Wistar rats weighing approximately 200 g were ovariectomized. Seven days after surgery half of the rats were injected intraperitoneally with suspensions of 17p-estradiol, the other half of the rats were used as controls and received only the vehicle (see below). The last injection of estradiol was given approximately 16 h (if not stated otherwise) before they were killed by decapitation.
Estrogen treatment The standard estrogen treatment of the rats consisted of daily intraperitoneal injections of 20 pg 17P-estradiol (dissolved in 1 ml NaCl with 1% bovine serum albumin (BSA)) for the indicated period of time. For time-course studies the rats were injected with a single intraperitoneal injection of 20 pg 17@estradiol and the rats were subsequently studied 6 and 24 h later; in addition, some of the rats were treated daily for 4 days before they were investigated.
Isolation of rat adipocytes The cells were prepared from the perirenal and parametrial fat pads of female Wistar rats as previously described (Richelsen and Pedersen, 1985). Minced adipose tissue was digested by collagenase (0.5 mg/ml) for 45 min at 37°C by gently shaking. After filtration and four washes the isolated adipocytes were resuspended in incubation buffer containing 10 mM Hepes, 2.5% human serum albumin (HSA), 1.35 mM NaCl, 4.8 mM KCl, 1.7 mM MgSO,, 2.5 mM CaCl,, 0.2 mM NaH,PO,, 1.0 mM Na,HPO, and 5 mM glucose (pH = 7.4 at 37°C). The size and concentration of adipocytes were determined as previously described (Richelsen and Pedersen, 1985).
15
Nuclear receptor assay 500 ~1 adipocyte suspension containing
about 10h cells were added to the tubes and incubated for 60 min at 37°C (in duplicate) with [“H]17Pestradiol at 3, 5, 10 and 15 nM in 10 ml polypropylene tubes (Spelsberg et al., 1987; Colvard et al., 1988; Pedersen et al., 1991). The binding reaction was terminated by addition of 5 ml of a cold (4°C) solution of 5 mM Hepes and 0.2 mM EDTA (pH = 7.4) to the tubes. The isolation of nuclei was performed by a slight modification of the method described by Spelsberg et al. (1987). Briefly, the adipocytes were centrifuged for 5 min at 800 x g at 4°C. The infranatant was removed, and the cells were resuspended in 2 ml cold 1 M sucrose solution (1 M sucrose, 10% glycerol, 0.1% Triton X-100, 50 mM Tris, 10 mM KCl, pH 7.4 at 4°C). The adipocytes were homogenized using a motor-driven Teflon pestle. The cell homogenates were carefully layered over 1 ml cold 1.4 M sucrose solution and centrifuged for 20 min at 6000 x g (4°C). The supernatant was removed and the nuclear pellet was resuspended in buffer containing 50 mM Tris, 10% glycerol (pH 7.4 at 4”C), filtered through a nitrocellulose filter, and finally counted in a p-counter. The non-specific binding was determined in the presence of 1 PM unlabeled estradiol. Specific binding was defined as total minus non-specific binding.
tosolic preparation was measured and the results are given as fmol estrogen bound/mg protein. Lipoprotein lipase activity
Lipoprotein lipase (LPL) activity was determined essentially as previously described (Hietanen and Greenwood, 1977). Briefly, the fat pads were homogenized and the homogenate was centrifuged. The LPL activity in the postmitochondrial supernatant was determined by estimating the specific hydrolysis of [‘4C]triolein. LPL activity was expressed as pmol of free fatty acid (FFA) released/h/mg protein. Insulin binding and lipolysis
The insulin binding was performed as previously described by incubating adipocytes for 120 min at 15°C together with [‘251]insulin (Pedersen et al., 1982). Lipolysis was determined by the release of glycerol as previously described (Richelsen et al., 1984). Calculations and statistics
Binding data were processed using the LIGAND program (Munson and Rodbard, 1980). Data are presented as mean f SEM. Significance was determined by using Student’s paired t-test. Results
Cytosolic estrogen receptor assay
Nuclear 17@estradiol binding
The assay was conducted essentially as previously described (Leake et al., 1981). Briefly, the adipose tissue was homogenized in a buffer containing 10 mM Tris-HCl, 1.5 mM EDTA and 10% glycerol, pH = 7.5 at 20°C and centrifuged for 40 min at 6000 X g (4°C). The supernatant below the fat cake was used and was incubated in triplicate with 15 nM 17/?-estradiol at 4°C for 18 h. The incubation was terminated by addition of dextran-coated charcoal (0.1% w/v charcoal and 0.01% w/v dextran T-70) and was subsequently centrifugated. 500 ~1 of the supernatant was transferred to scintillation tubes and counted. The non-specific binding was determined in the presence of 1 PM unlabeled estradiol. Specific binding was defined as total minus non-specific binding. Finally, the protein content in the cy-
Estrogen treatment for 4 days using intraperitoneal injections of 17&estradiol (20 pg) dissolved in saline enhanced the B,,, (the maximal binding capacity) for the nuclear estrogen receptor binding in isolated rat adipocytes from 3.82 f 0.27 fmol/106 cells to 12.7 f 5.0 fmol/106 cells (p < 0.05) (Table 1). In order to investigate the time which was necessary for estrogen to affect its own receptor binding we determined the nuclear receptor binding 6 and 24 h after a single injection of 20 pg 17P-estradiol. 6 h after a single injection the B,,, was increased from 3.82 _+0.27 fmol/106 cells to 9.8 _t 3.6 fmol/lO’ cells (p < 0.1) and 24 h after injection the B,,, was maximally increased to 12.7 + 5.5 fmol/106 cells (p < 0.05). The K, (the estrogen concentration giving half-maximal binding) was under all conditions
16 TABLE
1
EFFECTS OF ESTROGEN TREATMENT ON THE B,,, AND K, FOR THE NUCLEAR ESTROGEN RECEPTOR IN RAT ADIPOCYTES
* :
R,,;,, KJ Number
Control
6h
3.8kO.3 4.8+ 1.9 4
9.8k3.6 5.6k2.1 3
*
* p < 0.1; * * p < 0.05, compared
24 h
96 h
12.7k5.5 ** 2.9k 1.0 4
127~5.0 ** 3.9 + 2.3 4
with control
z
%
- LOG [INSULIN] (1.4)
Fig. 1. Effects of estrogen treatment on specific insulin binding in adipocytes. Adipocytes from control rats (empty circles) and from estrogen treated rats (filled circles) were incubated with 60 pmol/l [‘251]insulin at 15°C for 90 min in the presence of unlabeled insulin at the indicated concentrations. Data are given as mean+ SEM of five experiments. * p < 0.05; * * p < 0.01.
values.
unaltered by the estrogen treatment and was about 3-5 nM (Table 1). Both B,,, and K, were calculated from the binding data by using the LIGAND computer program developed by Munson and Rodbard (1980).
adipocytes. The specific insulin binding was almost doubled in adipocytes from estrogen treated rats (20 pg estradiol in NaCl every day for 7 days) compared to control rats: 21.7 + 3.0 and 10.4 + 2.1 insulin-binding percent/ lo6 adipocytes ( p < 0.011, respectively (at an insulin tracer concentration of 60 PM). Displacement studies revealed that the ED,, was similar in both groups: 3.3 f 0.5 nM and 2.4 _t 0.7 nM, respectively (Fig. 1). Time-course studies revealed that already 6 h after a single estrogen injection (20 pg estradiol in NaCl) the specific insulin binding was increased by 78 &-7% (p < 0.05) (Table 2) and 24 h after a single injection the binding was increased by 115 f 5% (p < 0.01). When the rats were treated every day for 4 days (20 pg estradiol daily) the specific insulin binding was increased by 149 _t 6% (p < 0.001) and the binding was not
Cytosolic estrogen receptor binding
Estrogen treatment (20 pg estradiol in NaCl every day for 4 days) tended to increase the specific cytosolic estrogen binding by 20% from 4.15 f 0.39 fmol estrogen bound/mg protein to 4.96 I~I1.1 fmol estrogen bound/mg protein (investigated at a estrogen tracer concentration of 15 nM; n = 6; however, the increase was not statistically significant. Insulin binding
The effects of estrogen treatment were also investigated on the insulin binding in isolated
TABLE
E:
is--- *
Rats were treated with estradiol intraperitoneally (20 pg estradiol dissolved in saline) and the rats were investigated 6 and 24 h later. Four of the rats were injected daily (20 pg estradiol dissolved in saline) for 4 days, before they were used. The isolated adipocytes were incubated with [‘H]17P-estradiol at 3. 5, 10 and 15 nM, for 60 min. The E,,, and K, were estimated according to Scatchard.
2
TIME-COURSE
STUDY
OF SPECIFIC
INSULIN
BINDING
IN RAT ADIPOCYTES
Rats were treated with estradiol intraperitoneally (20 pg estradiol dissolved in saline) and the rats were investigated 6 and 24 h later. Some of the rats were injected daily (20 pg estradiol dissolved in saline) for 4 days and 7 days, respectively, before they were used. The isolated adipocytes were incubated with 60 pM [1251]insulin at 15°C for 120 min.
Insulin binding Number
percent
of control
Control
6h
100 4
178k7 4
* p < 0.05; * * p < 0.01; * * * p < 0.001, compared
with control
24 h *
values.
215+6 4
96 h **
249k6 4
168 h ***
220+8 4
**
17 TABLE
3
EFFECTS
OF ESTROGEN
ON BODY
WEIGHT,
ADIPOCYTE
The body weight, adipocyte size and LPL activity in adipocytes estrogen in saline every day for 7 days).
Body weight (g) Adipocyte size ( x 10 I2 1) LPL activity prnol FFA/h/mg
protein)
in control
LIPASE
ACTIVITY
rats and in estrogen
treated
rats (20 ~g
n
Estrogen
n
Significance
225.1 * 3.7 171.4 + 16.1 2.11? 0.46
12 12 7
209.6 k 2.7 125.3 k 14.3 0.x0* 0.22
12 12 7
p < 0.01 p < 0.01 p < 0.05
was ex-
Metabolic effects of estrogen treatment
The control rats had a weight gain of approximately 11% in 7 days (22 g> whereas estrogen treated rats (20 pug estradiol every day) gained about 4% in the same period (p < 0.05). Adipocytes were significantly larger in control rats (p < 0.01) compared to adipocytes from estrogen substituted rats (Table 3). Furthermore, the administration of estrogen resulted in a significant decrease in lipoprotein lipase activity in adipose tissue from 2.11 f 0.46 pmol FFA re-
:
1
leased/h/mg protein to 0.80 + 0.22 pmol FFA released/h/mg protein ( p < 0.051, corresponding to a decrease in LPL activity by 62% (Table 3). The glycerol release in adipocytes from rats with or without estrogen substitution was equal under basal conditions (no stimulation). However, in adipocytes from estrogen treated rats (20 pg estradiol in NaCl every day for 4 days) the glycerol release was significantly increased by stimulation with adenosine deaminase (ADA) alone or ADA combined with epinephrine compared with control rats (Fig. 2). Thus, a significant increase in the lipolytic response was found in adipocytes from estrogen treated rats.
Discussion
1400.
w ;
LIPOPROTEIN
Control
further increased when the treatment tended to 7 days (Table 2).
1600
SIZE AND
were investigated
~1200.
d [Lu) lOOO10 g 5 600. 8 E Flc
600-
o
400-
-
*DA:
EPI:
-
+
-
-
6.0
f
+
-LOG
[EPINEPHRINE)
7.5
+
7.0
+
6.5
+
6.0
+
5.5
(MoL)
Fig. 2. Effects of estrogen on epinephrine stimulated lipolysis in rat adipocytes. Adipocytes from control rats (empty circles) and from estrogen treated (20 pg estrogen dissolved in saline given every day for 7 days) (filled circles) were incubated under both basal and stimulated conditions (ADA 0.5 U/ml and the indicated concentration of epinephrine) for 60 min at 37°C. Data are given as glycerol released per lo6 cells, mean + SEM of five experiments. * p < 0.05; * * p < 0.01.
Estrogen treatment of rats has been shown to increase the lipolytic response triggered by catecholamines (Benoit et al., 1982; Pasquier et al., 1988) and to decrease the lipoprotein lipase activity (Gray and Wade, 1980; Steingrimsdottir et al., 1980; Rebuff&Strive, 1987a) as also shown in the present paper. These metabolic alterations might result in increased release of free fatty acids (FFA) and decreased triglyceride assimilation. The result will be smaller adipocytes and decreased body fat content. Thus, these findings emphasize the great biological effect of estrogen on adipose tissue and adipocyte metabolism. Estrogen treatment in humans has also been shown to alter the fat distribution (Vague et al., 1984) and adipocyte size (Krotkiewski and Bjiirntorp, 1978). Furthermore, adipocytes from different fat
depots have different metabolisms, the factors responsible for these site differences are still unknown, but steroids including estradiol has been suggested to play a role (Rebuff&Strive et al., 1985; Rebuff&Strive, 1987b; Bjiirntorp, 1988; Bjorntorp et al., 1990). In a recent study we demonstrated that rat adipocytes from different fat depots had different numbers of nuclear estrogen receptors (Pedersen et al., 1991). The nuclear receptor assay we used has been shown to mimic the in vivo patterns of nuclear binding and to correlate with steroid regulation of gene transcription in animal mode1 systems (Spelsberg et al., 1987). This is achieved by preserving the normal biochemical processes and microbiology of the cells via incubating intact cells with the labeled steroid and by avoiding loss of receptors by exposure of proteases because intracellular organelles such as lysosomes are intact during the steroid incubation Wmehara et al., 1988). Therefore, by using this technique it has been possible to detect minute regulation of the nuclear estradiol receptor binding caused by hormonal manipulation in isolated adipocytes (Pedersen et al., 1991). Treating rats with estrogen increased the nuclear estradiol receptor binding in isolated adipocytes. These experiments comply very well with the data of Shapiro et al. (1989). They demonstrated that a single estrogen injection could induce both the mRNA for the estrogen receptor and the number of estrogen receptors. Furthermore, Beckman et al. (1989) found that the nuclear estradiol receptor number in rat uterus was increased after a single estradiol injection. Finally, De Pegola et al. (1991) demonstrated that androgen treatment of rat adipose precursor cells in culture up-regulates the nuclear androgen receptor number in these cells. In order to investigate whether the increased nuclear estrogen receptor binding was due to a redistribution of the estrogen receptor, we measured the cytosolic estrogen receptor as well. These studies demonstrated that there was a tendency for increased cytosolic estrogen receptor content as well. Thus, the present study indicates that estrogen treatment clearly may induce its own receptor in rat adipocytes. Earlier studies have demonstrated that treatment of rats with slowly released
estradiol (i.e. dissolved in oil) was accompanied by a reduced number of cy~usolic estrogen receptors in adipocytes (Gray and Wade, 1979; Rebuff&Strive, 1987a). In addition, we investigated the effects of slowly released estrogen on the nuclear estrogen receptor binding, and found the nuclear estrogen receptor binding reduced to a similar extent (data not shown). The reason for the reduced binding is probably that most of the estrogen receptors are occupied because of the sustained high concentration of estrogen after treatment with slowly released estrogen (Stone, 1963). Furthermore, estrogen treatment increased the insulin binding 6 h after a single injection of 17&estradiol, and maximal induced insulin binding was observed in adipocytes from rats treated daily for 4 days. The mechanisms behind the increased insulin receptor binding are presently unknown. However, estrogen may have direct effect on the adipocytes, as Ryan and Enns (1988) also observed in adipocytes in culture that estrogen almost doubled the insulin receptor binding after incubation for 72 h. In addition, Ballejo et al. (1983) have demonstrated increased insulin receptor binding in adipocytes from estrogen treated rats. In conclusion, the present study revealed that estrogen treatment of ovariectomized rats had a positive feedback effect on its own nuclear receptor and, thus, increased the number of nuclear estrogen receptors; furthermore, there was a tendency for increased cytosolic estrogen receptor content as well. In addition, the number of insulin receptors in adipocytes was increased by estrogen treatment, whereas the affinity of the two receptors was unaltered. Besides its effects on estrogen and insulin receptors estrogen was found to increase the lipolytic response, decrease LPL activity and diminish adipocyte cell size. There is indirect evidence to suggest that estrogens play a role in regional adipose tissue distribution. In this respect the alterations of adipose tissue distribution in relation to puberty and menopause seem of particular interest. Thus, the finding that estrogen and other hormones may regulate the nuclear estrogen receptor binding may be important for adipose tissue distribution and metabolism in man.
19
Acknowledgments
We appreciate the technical assistance of J. Soholt, L. Trudso, L. Pedersen and P. Sonne. This study was supported by the Novo Foundation, the Nordic Insulin Foundation, Aarhus University, A.P. Moller Foundation, the Danish Diabetes Association, the Jenny Vissing Foundation and the Danish Medical Research Council.
References Ackerman, G.E., MacDonald, P.C., Gudelsky, G., Mendelson, C.R. and Simpson, E.R. (1981) Endocrinology 109, 20842088. Ballejo, G., Saleem, T.H., Khan-Dawood, F.S., Tsibris, J.C.M. and Spellacy, W.N. (1983) Contraception 28, 413-422. Beckman, W.C., Hudspeth, D.A., Golding, T., Hubbert, L., Akkerman, J. and Korach, K.S. (1989) Endocrinology 124, 2651-2658. Benoit, V., Valette, A., Mercier, L., Meignen, J.M. and Boyer, J. (1982) Biochem. Biophys. Res. Commun. 109, 11861191. Bjorntorp, P. (1988) Acta Med. Stand. Suppl. 723, 121-134. Bjorntorp, P., Ottosen, M., Rebuff&Strive, M. and Xu, X. (1990) in Obesity: Towards a Molecular Approach (Bray, G.A., Ricquier, D. and Spigelman, B.M., eds.), pp. 147157, Wiley-Liss, New York. Boyd-Leinen, P.A., Fournier, D. and Spelsberg, T.C. (1982) Endocrinology 111, 30-36. Colvard, D.S., Jankus, W.R., Berg, N.J., Graham, M.L., Jiang, N.-S., Ingle, J.N. and Spelsberg, T.C. (1988) Clin. Chem. 34, 363-369. De Pegola, G., Xu, X., Yang, S., Giorgino, R. and Bjorntorp, P. (1991) Int. J. Obesity 15, 54-54. Freedman, D.S., Jacobsen, J.S., Barboriak, J.J., Sobocinski, K.A., Anderson, A.J., Kissebah, A.H., Sasse, E.A. and Gruchow, H.W. (1990) Circulation 81, 1498-1506. Fried, S.K. and Kral, J.G. (1987) Int. J. Obesity 11, 129-140. Gehring, U. and Tomkins, G.M. (1974) Cell 3, 301-306. Gray, J.M. and Wade, G.N. (1979) Endocrinology 104, 13771382. Gray. J.M. and Wade, G.N. (1980) Am. J. Physiol. 239, E237-E241. Gray, J.M., Dudley, S.D. and Wade, G.N. (19811 Am. J. Physiol. 240, E43-E46.
Hietanen, E. and Greenwood, M.C.R. (1977) J. Lipid Res. 18, 480-490. Krotkiewski, M. and Bjorntorp, P. (1978) J. Endocrinol. Invest. 1, 364-371. Krotkiewski, M., Bjorntorp, P., Sjostrom, L. and Smith, U. (1983) J. Clin. Invest. 72, 1150-1162. Leake, R.E., Laing, L., Calman, K.C., MacBeth, F.R., Crawford, D. and Smith, D.C. (1981) Br. J. Cancer 43, 59-65. Lundgreen, H., Bengtsson, C., Blohme, G., Lapidus, L. and SjGstrGm, L. (1989) Int. J. Obesity 13, 413-423. MacFarlane, J.K., Fleiszer, D. and Fazekas, A.G. (1980) Cancer 45, 2998-3003. Munson, P.J. and Rodbard, D. (1980) Anal. Biochem. 107, 220-239. Pasquier, Y.-N., Pecquery, R. and Guidicelli, Y. (1988) Biochem. Biophys. Res. Commun. 154, 1151-1159. Pecquery, R., Leneveu, M.C and Giudicelli, Y. (1986) Endocrinology 118, 2210-2216. Pedersen, O., Hjollund, E. and Lindskov, H.O. (1982) Am. J. Physiol. 243, E158-E167. Pedersen, S.B., Borglum, J.D., Eriksen, E.F. and Richelsen, B. (1991) Biochim. Biophys. Acta 1093, 80-86. Rebuff&Strive, M. (1987a) Acta Physiol. Stand. 129, 471-477. Rebuff&Strive, M. (1987bI Acta Med. Stand. Suppl. 723, 143-146. Rebuff&Strive, M., Enk, L., Crona, N., L&troth, P., Abrahamsson, L., Smith, U. and Bjorntorp, P. (1985) J. Clin. Invest. 75, 1973-1976. Rebuff&Strive, M., Bronnegard, M., Nilsson, A., Eldh, J., Gustafsson, J.-A. and Bjorntorp, P. (1990) J. Clin. Endocrinol. Metab. 71, 1215-1219. Richelsen, B. and Pedersen, 0. (1985) Endocrinology 116, 1182-1187. Richelsen. B., Eriksen, E.F., Beck-Nielsen, H. and Pedersen, 0. (1984) J. Clin. Endocrinol. Metab. 59, 7-12. Ryan, E.A. and Enns, L. (1988) J. Clin. Endocrinol. Metab. 67, 341-347. Shapiro, D.J., Barton, M.C., Mckearin, D.M. et al. (1989) Recent Prog. Horm. Res. 4.5, 29-58. Spelsberg, T.C., Graham, M.K., Berg, N.J., Umehara, T., Riehl, E., Coulam, C.B. and Ingle, J.N. (1987) Endocrinology 121, 631-644. Steingrimsdottir, L., Brasel, J. and Greenwood, M.R.C. (1980) Am. J. Physiol. 239, E162-E167. Stone, G.M. (1963) J. Endocrinol. 27, 281-288. Umehara, T., Graham, M.L., Berg, N.J., Lieber, M.M. and Spelsberg, T.C. (1988) J. Steroid Biochem. 31, 15-25. Vague. J., Meignen, J.M. and Negrin, J.F. (1984) Worm. Metab. Res. 16, 380-381. Wade, G.N. and Gray, J.M. (1978) Endocrinology 103, 16951701.
MOLCEL
Scientific
Publishers
Ireland,
13
Ltd. 0303.7207/92/$0.5.00
02736
Effects of in vivo estrogen treatment on adipose tissue metabolism and nuclear estrogen receptor binding in isolated rat adipocytes Steen B. Pedersen, Jens D. Borglum, Torben Moller-Pedersen
and Bjarn Richelsen
l’nicersity Clinic of Endocrinology and Internal Medicine, Aarhus Amtssygehus, Tage Hansensgade, DK-8000 Aarhus C, Denmark (Received
Key words: Estrogen;
Adipose
6 December
tissue metabolism;
Nuclear
1991; accepted
estrogen
receptor
27 December
binding;
1991)
Adipocyte;
(Rat)
Summary We have previously demonstrated the existence of nuclear estrogen receptors in isolated adipocytes (Pedersen et al. (1991) Biochim. Biophys. Acta 1093, 80-86). In the present study we have investigated the regulatory properties of these nuclear estrogen receptors, in addition to the metabolic effects of estrogen on adipose tissue metabolism. Estrogen treatment (20 pg 17/Sestradiol in NaCl for 7 days) decreased lipoprotein lipase activity (LPL) in the adipose tissue by 62% (p < 0.051, decreased adipocyte size by 27% (p < 0.01) and diminished the normal postovariectomy weight gain. Furthermore, estrogen treatment increased the nuclear estrogen receptor binding in adipocytes; in addition, there was a tendency for increased cytosolic estrogen receptor content as well. Time course studies revealed that already 6 h after a single estrogen injection the B,,, increased from 3.82 k 0.3 fmol/106 cells to 9.8 f 3.6 fmol/106 cells (p < 0.1) and 24 h after a single injection the B,,, was maximally increased to 12.7 k 5.5 fmol/lO’ cells (p < 0.05). The K, was similar at all time points (about 3-5 nM). Furthermore, the specific insulin receptor binding was increased in adipocytes from estrogen treated rats. The specific insulin binding was maximally increased by 149 f 6% (p < 0.001) after 4 days of daily estrogen injections. The increased binding seemed to be due to an increased number of insulin receptors on adipocytes from estrogen treated rats with no alteration of the ED,, value. In conclusion it was found that estrogen treatment has a positive feedback effect on its own nuclear receptor. In addition, estrogen treatment was found to have several metabolic effects in the adipose tissue. Estrogen treatment increased the insulin receptor number, decreased LPL activity, increased the lipolytic response in adipocytes and finally decreased body weight and adipocyte cell size.
Introduction The health consequences of obesity have been correlated with the accumulation of adipose tis-
Correspondence to: Steen B. Pedersen, University Clinic of Endocrinology and Internal Medicine, Aarhus Amtssygebus, Tage Hansensgade, DK-8000 Aarhus C, Denmark.
sue in the abdominal region (android type of obesity), especially accumulation of intraabdominal adipose tissue, whereas the accumulation of adipose tissue in the gluteal and femoral regions (gynoid type) has been considered much less hazardous (Bjiirntorp, 1988; Lundgreen et al., 1989). The factors responsible for the distribution of adipose tissue are mostly unknown but the distribution is in some way sexually linked (Krotkiew-
14
ski et al., 1983; Fried and Kral, 1987; Freedman et al., 1990). Furthermore, estrogen treatment has been shown to affect both the fat distribution (Vague et al., 1984) and the metabolism of isolated fat cells (Ackerman et al., 1981; Benoit et al., 1982; Pecquery et al., 1986; Rebuff&Strive, 1987a; Pasquier et al., 1988). Whether the effects of estrogen on the adipose tissue are mediated by direct or indirect mechanisms is presently unknown (Rebuff&&rive et al., 1990). However, we and other groups have found estrogen receptors in isolated adipocytes, either cytoplasmic receptors (Wade and Gray, 1978; Gray and Wade, 1980; Rebuff&Strive, 1987a) or nuclear receptors (Gray et al., 1981; Pedersen et al., 1991). Thus, it may be suggested that estrogen may have direct effects on the adipocytes through these receptors. Studies in other tissues have demonstrated the existence of non-functional steroid receptors (Gehring and Tomkins, 1974; Boyd-Leinen et al., 1982; Spelsberg et al., 1987; Colvard et al., 19881, i.e. cytoplasmic receptors that are able to bind the steroid but unable to become activated and tightly bound to the nucleus. Thus, methods based on receptor assay after lysis of the cell may yield results that do not correlate to biological effects of the steroid in the given cell type (MacFarlane et al., 1980). Recently, methods have become available that quantitate the number of functional receptors (Spelsberg et al., 1987; Colvard et al., 1988). In a recent study we found that the nuclear estrogen binding in isolated adipocytes could be modulated by various hormones such as catecholamines and insulin (Pedersen et al., 1991). In order to get more insight in the factors which regulate the number and the affinity of the nuclear estrogen receptor we investigated the effects of estrogen treatment on these parameters. In addition we investigated the effect of estrogen treatment on the metabolism of the adipose tissue (lipoprotein lipase activity, insulin receptor binding and lipolytic activity). Materials
and methods
Chemicals 17@[2,4,6,7,16,17-“HlEstradiol, 140-170 Ci/mmol, was obtained
spec. act. = from Amersham
(UK). [Carboxy/-‘“ClTriolein, spec. act. = 112 mCi/mmol, was purchased from NEN Research Products and [12sI]insulin was kindly donated by Novo-Nordisk Research Institute. Crude collagenase from Clostridium histeolyticum was obtained from Worthington Biochemical Corp., Freehold, NJ, USA. Estradiol benzoate and sesame oleum were obtained as pharmaceutical. All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Animals Female Wistar rats weighing approximately 200 g were ovariectomized. Seven days after surgery half of the rats were injected intraperitoneally with suspensions of 17p-estradiol, the other half of the rats were used as controls and received only the vehicle (see below). The last injection of estradiol was given approximately 16 h (if not stated otherwise) before they were killed by decapitation.
Estrogen treatment The standard estrogen treatment of the rats consisted of daily intraperitoneal injections of 20 pg 17P-estradiol (dissolved in 1 ml NaCl with 1% bovine serum albumin (BSA)) for the indicated period of time. For time-course studies the rats were injected with a single intraperitoneal injection of 20 pg 17@estradiol and the rats were subsequently studied 6 and 24 h later; in addition, some of the rats were treated daily for 4 days before they were investigated.
Isolation of rat adipocytes The cells were prepared from the perirenal and parametrial fat pads of female Wistar rats as previously described (Richelsen and Pedersen, 1985). Minced adipose tissue was digested by collagenase (0.5 mg/ml) for 45 min at 37°C by gently shaking. After filtration and four washes the isolated adipocytes were resuspended in incubation buffer containing 10 mM Hepes, 2.5% human serum albumin (HSA), 1.35 mM NaCl, 4.8 mM KCl, 1.7 mM MgSO,, 2.5 mM CaCl,, 0.2 mM NaH,PO,, 1.0 mM Na,HPO, and 5 mM glucose (pH = 7.4 at 37°C). The size and concentration of adipocytes were determined as previously described (Richelsen and Pedersen, 1985).
15
Nuclear receptor assay 500 ~1 adipocyte suspension containing
about 10h cells were added to the tubes and incubated for 60 min at 37°C (in duplicate) with [“H]17Pestradiol at 3, 5, 10 and 15 nM in 10 ml polypropylene tubes (Spelsberg et al., 1987; Colvard et al., 1988; Pedersen et al., 1991). The binding reaction was terminated by addition of 5 ml of a cold (4°C) solution of 5 mM Hepes and 0.2 mM EDTA (pH = 7.4) to the tubes. The isolation of nuclei was performed by a slight modification of the method described by Spelsberg et al. (1987). Briefly, the adipocytes were centrifuged for 5 min at 800 x g at 4°C. The infranatant was removed, and the cells were resuspended in 2 ml cold 1 M sucrose solution (1 M sucrose, 10% glycerol, 0.1% Triton X-100, 50 mM Tris, 10 mM KCl, pH 7.4 at 4°C). The adipocytes were homogenized using a motor-driven Teflon pestle. The cell homogenates were carefully layered over 1 ml cold 1.4 M sucrose solution and centrifuged for 20 min at 6000 x g (4°C). The supernatant was removed and the nuclear pellet was resuspended in buffer containing 50 mM Tris, 10% glycerol (pH 7.4 at 4”C), filtered through a nitrocellulose filter, and finally counted in a p-counter. The non-specific binding was determined in the presence of 1 PM unlabeled estradiol. Specific binding was defined as total minus non-specific binding.
tosolic preparation was measured and the results are given as fmol estrogen bound/mg protein. Lipoprotein lipase activity
Lipoprotein lipase (LPL) activity was determined essentially as previously described (Hietanen and Greenwood, 1977). Briefly, the fat pads were homogenized and the homogenate was centrifuged. The LPL activity in the postmitochondrial supernatant was determined by estimating the specific hydrolysis of [‘4C]triolein. LPL activity was expressed as pmol of free fatty acid (FFA) released/h/mg protein. Insulin binding and lipolysis
The insulin binding was performed as previously described by incubating adipocytes for 120 min at 15°C together with [‘251]insulin (Pedersen et al., 1982). Lipolysis was determined by the release of glycerol as previously described (Richelsen et al., 1984). Calculations and statistics
Binding data were processed using the LIGAND program (Munson and Rodbard, 1980). Data are presented as mean f SEM. Significance was determined by using Student’s paired t-test. Results
Cytosolic estrogen receptor assay
Nuclear 17@estradiol binding
The assay was conducted essentially as previously described (Leake et al., 1981). Briefly, the adipose tissue was homogenized in a buffer containing 10 mM Tris-HCl, 1.5 mM EDTA and 10% glycerol, pH = 7.5 at 20°C and centrifuged for 40 min at 6000 X g (4°C). The supernatant below the fat cake was used and was incubated in triplicate with 15 nM 17/?-estradiol at 4°C for 18 h. The incubation was terminated by addition of dextran-coated charcoal (0.1% w/v charcoal and 0.01% w/v dextran T-70) and was subsequently centrifugated. 500 ~1 of the supernatant was transferred to scintillation tubes and counted. The non-specific binding was determined in the presence of 1 PM unlabeled estradiol. Specific binding was defined as total minus non-specific binding. Finally, the protein content in the cy-
Estrogen treatment for 4 days using intraperitoneal injections of 17&estradiol (20 pg) dissolved in saline enhanced the B,,, (the maximal binding capacity) for the nuclear estrogen receptor binding in isolated rat adipocytes from 3.82 f 0.27 fmol/106 cells to 12.7 f 5.0 fmol/106 cells (p < 0.05) (Table 1). In order to investigate the time which was necessary for estrogen to affect its own receptor binding we determined the nuclear receptor binding 6 and 24 h after a single injection of 20 pg 17P-estradiol. 6 h after a single injection the B,,, was increased from 3.82 _+0.27 fmol/106 cells to 9.8 _t 3.6 fmol/lO’ cells (p < 0.1) and 24 h after injection the B,,, was maximally increased to 12.7 + 5.5 fmol/106 cells (p < 0.05). The K, (the estrogen concentration giving half-maximal binding) was under all conditions
16 TABLE
1
EFFECTS OF ESTROGEN TREATMENT ON THE B,,, AND K, FOR THE NUCLEAR ESTROGEN RECEPTOR IN RAT ADIPOCYTES
* :
R,,;,, KJ Number
Control
6h
3.8kO.3 4.8+ 1.9 4
9.8k3.6 5.6k2.1 3
*
* p < 0.1; * * p < 0.05, compared
24 h
96 h
12.7k5.5 ** 2.9k 1.0 4
127~5.0 ** 3.9 + 2.3 4
with control
z
%
- LOG [INSULIN] (1.4)
Fig. 1. Effects of estrogen treatment on specific insulin binding in adipocytes. Adipocytes from control rats (empty circles) and from estrogen treated rats (filled circles) were incubated with 60 pmol/l [‘251]insulin at 15°C for 90 min in the presence of unlabeled insulin at the indicated concentrations. Data are given as mean+ SEM of five experiments. * p < 0.05; * * p < 0.01.
values.
unaltered by the estrogen treatment and was about 3-5 nM (Table 1). Both B,,, and K, were calculated from the binding data by using the LIGAND computer program developed by Munson and Rodbard (1980).
adipocytes. The specific insulin binding was almost doubled in adipocytes from estrogen treated rats (20 pg estradiol in NaCl every day for 7 days) compared to control rats: 21.7 + 3.0 and 10.4 + 2.1 insulin-binding percent/ lo6 adipocytes ( p < 0.011, respectively (at an insulin tracer concentration of 60 PM). Displacement studies revealed that the ED,, was similar in both groups: 3.3 f 0.5 nM and 2.4 _t 0.7 nM, respectively (Fig. 1). Time-course studies revealed that already 6 h after a single estrogen injection (20 pg estradiol in NaCl) the specific insulin binding was increased by 78 &-7% (p < 0.05) (Table 2) and 24 h after a single injection the binding was increased by 115 f 5% (p < 0.01). When the rats were treated every day for 4 days (20 pg estradiol daily) the specific insulin binding was increased by 149 _t 6% (p < 0.001) and the binding was not
Cytosolic estrogen receptor binding
Estrogen treatment (20 pg estradiol in NaCl every day for 4 days) tended to increase the specific cytosolic estrogen binding by 20% from 4.15 f 0.39 fmol estrogen bound/mg protein to 4.96 I~I1.1 fmol estrogen bound/mg protein (investigated at a estrogen tracer concentration of 15 nM; n = 6; however, the increase was not statistically significant. Insulin binding
The effects of estrogen treatment were also investigated on the insulin binding in isolated
TABLE
E:
is--- *
Rats were treated with estradiol intraperitoneally (20 pg estradiol dissolved in saline) and the rats were investigated 6 and 24 h later. Four of the rats were injected daily (20 pg estradiol dissolved in saline) for 4 days, before they were used. The isolated adipocytes were incubated with [‘H]17P-estradiol at 3. 5, 10 and 15 nM, for 60 min. The E,,, and K, were estimated according to Scatchard.
2
TIME-COURSE
STUDY
OF SPECIFIC
INSULIN
BINDING
IN RAT ADIPOCYTES
Rats were treated with estradiol intraperitoneally (20 pg estradiol dissolved in saline) and the rats were investigated 6 and 24 h later. Some of the rats were injected daily (20 pg estradiol dissolved in saline) for 4 days and 7 days, respectively, before they were used. The isolated adipocytes were incubated with 60 pM [1251]insulin at 15°C for 120 min.
Insulin binding Number
percent
of control
Control
6h
100 4
178k7 4
* p < 0.05; * * p < 0.01; * * * p < 0.001, compared
with control
24 h *
values.
215+6 4
96 h **
249k6 4
168 h ***
220+8 4
**
17 TABLE
3
EFFECTS
OF ESTROGEN
ON BODY
WEIGHT,
ADIPOCYTE
The body weight, adipocyte size and LPL activity in adipocytes estrogen in saline every day for 7 days).
Body weight (g) Adipocyte size ( x 10 I2 1) LPL activity prnol FFA/h/mg
protein)
in control
LIPASE
ACTIVITY
rats and in estrogen
treated
rats (20 ~g
n
Estrogen
n
Significance
225.1 * 3.7 171.4 + 16.1 2.11? 0.46
12 12 7
209.6 k 2.7 125.3 k 14.3 0.x0* 0.22
12 12 7
p < 0.01 p < 0.01 p < 0.05
was ex-
Metabolic effects of estrogen treatment
The control rats had a weight gain of approximately 11% in 7 days (22 g> whereas estrogen treated rats (20 pug estradiol every day) gained about 4% in the same period (p < 0.05). Adipocytes were significantly larger in control rats (p < 0.01) compared to adipocytes from estrogen substituted rats (Table 3). Furthermore, the administration of estrogen resulted in a significant decrease in lipoprotein lipase activity in adipose tissue from 2.11 f 0.46 pmol FFA re-
:
1
leased/h/mg protein to 0.80 + 0.22 pmol FFA released/h/mg protein ( p < 0.051, corresponding to a decrease in LPL activity by 62% (Table 3). The glycerol release in adipocytes from rats with or without estrogen substitution was equal under basal conditions (no stimulation). However, in adipocytes from estrogen treated rats (20 pg estradiol in NaCl every day for 4 days) the glycerol release was significantly increased by stimulation with adenosine deaminase (ADA) alone or ADA combined with epinephrine compared with control rats (Fig. 2). Thus, a significant increase in the lipolytic response was found in adipocytes from estrogen treated rats.
Discussion
1400.
w ;
LIPOPROTEIN
Control
further increased when the treatment tended to 7 days (Table 2).
1600
SIZE AND
were investigated
~1200.
d [Lu) lOOO10 g 5 600. 8 E Flc
600-
o
400-
-
*DA:
EPI:
-
+
-
-
6.0
f
+
-LOG
[EPINEPHRINE)
7.5
+
7.0
+
6.5
+
6.0
+
5.5
(MoL)
Fig. 2. Effects of estrogen on epinephrine stimulated lipolysis in rat adipocytes. Adipocytes from control rats (empty circles) and from estrogen treated (20 pg estrogen dissolved in saline given every day for 7 days) (filled circles) were incubated under both basal and stimulated conditions (ADA 0.5 U/ml and the indicated concentration of epinephrine) for 60 min at 37°C. Data are given as glycerol released per lo6 cells, mean + SEM of five experiments. * p < 0.05; * * p < 0.01.
Estrogen treatment of rats has been shown to increase the lipolytic response triggered by catecholamines (Benoit et al., 1982; Pasquier et al., 1988) and to decrease the lipoprotein lipase activity (Gray and Wade, 1980; Steingrimsdottir et al., 1980; Rebuff&Strive, 1987a) as also shown in the present paper. These metabolic alterations might result in increased release of free fatty acids (FFA) and decreased triglyceride assimilation. The result will be smaller adipocytes and decreased body fat content. Thus, these findings emphasize the great biological effect of estrogen on adipose tissue and adipocyte metabolism. Estrogen treatment in humans has also been shown to alter the fat distribution (Vague et al., 1984) and adipocyte size (Krotkiewski and Bjiirntorp, 1978). Furthermore, adipocytes from different fat
depots have different metabolisms, the factors responsible for these site differences are still unknown, but steroids including estradiol has been suggested to play a role (Rebuff&Strive et al., 1985; Rebuff&Strive, 1987b; Bjiirntorp, 1988; Bjorntorp et al., 1990). In a recent study we demonstrated that rat adipocytes from different fat depots had different numbers of nuclear estrogen receptors (Pedersen et al., 1991). The nuclear receptor assay we used has been shown to mimic the in vivo patterns of nuclear binding and to correlate with steroid regulation of gene transcription in animal mode1 systems (Spelsberg et al., 1987). This is achieved by preserving the normal biochemical processes and microbiology of the cells via incubating intact cells with the labeled steroid and by avoiding loss of receptors by exposure of proteases because intracellular organelles such as lysosomes are intact during the steroid incubation Wmehara et al., 1988). Therefore, by using this technique it has been possible to detect minute regulation of the nuclear estradiol receptor binding caused by hormonal manipulation in isolated adipocytes (Pedersen et al., 1991). Treating rats with estrogen increased the nuclear estradiol receptor binding in isolated adipocytes. These experiments comply very well with the data of Shapiro et al. (1989). They demonstrated that a single estrogen injection could induce both the mRNA for the estrogen receptor and the number of estrogen receptors. Furthermore, Beckman et al. (1989) found that the nuclear estradiol receptor number in rat uterus was increased after a single estradiol injection. Finally, De Pegola et al. (1991) demonstrated that androgen treatment of rat adipose precursor cells in culture up-regulates the nuclear androgen receptor number in these cells. In order to investigate whether the increased nuclear estrogen receptor binding was due to a redistribution of the estrogen receptor, we measured the cytosolic estrogen receptor as well. These studies demonstrated that there was a tendency for increased cytosolic estrogen receptor content as well. Thus, the present study indicates that estrogen treatment clearly may induce its own receptor in rat adipocytes. Earlier studies have demonstrated that treatment of rats with slowly released
estradiol (i.e. dissolved in oil) was accompanied by a reduced number of cy~usolic estrogen receptors in adipocytes (Gray and Wade, 1979; Rebuff&Strive, 1987a). In addition, we investigated the effects of slowly released estrogen on the nuclear estrogen receptor binding, and found the nuclear estrogen receptor binding reduced to a similar extent (data not shown). The reason for the reduced binding is probably that most of the estrogen receptors are occupied because of the sustained high concentration of estrogen after treatment with slowly released estrogen (Stone, 1963). Furthermore, estrogen treatment increased the insulin binding 6 h after a single injection of 17&estradiol, and maximal induced insulin binding was observed in adipocytes from rats treated daily for 4 days. The mechanisms behind the increased insulin receptor binding are presently unknown. However, estrogen may have direct effect on the adipocytes, as Ryan and Enns (1988) also observed in adipocytes in culture that estrogen almost doubled the insulin receptor binding after incubation for 72 h. In addition, Ballejo et al. (1983) have demonstrated increased insulin receptor binding in adipocytes from estrogen treated rats. In conclusion, the present study revealed that estrogen treatment of ovariectomized rats had a positive feedback effect on its own nuclear receptor and, thus, increased the number of nuclear estrogen receptors; furthermore, there was a tendency for increased cytosolic estrogen receptor content as well. In addition, the number of insulin receptors in adipocytes was increased by estrogen treatment, whereas the affinity of the two receptors was unaltered. Besides its effects on estrogen and insulin receptors estrogen was found to increase the lipolytic response, decrease LPL activity and diminish adipocyte cell size. There is indirect evidence to suggest that estrogens play a role in regional adipose tissue distribution. In this respect the alterations of adipose tissue distribution in relation to puberty and menopause seem of particular interest. Thus, the finding that estrogen and other hormones may regulate the nuclear estrogen receptor binding may be important for adipose tissue distribution and metabolism in man.
19
Acknowledgments
We appreciate the technical assistance of J. Soholt, L. Trudso, L. Pedersen and P. Sonne. This study was supported by the Novo Foundation, the Nordic Insulin Foundation, Aarhus University, A.P. Moller Foundation, the Danish Diabetes Association, the Jenny Vissing Foundation and the Danish Medical Research Council.
References Ackerman, G.E., MacDonald, P.C., Gudelsky, G., Mendelson, C.R. and Simpson, E.R. (1981) Endocrinology 109, 20842088. Ballejo, G., Saleem, T.H., Khan-Dawood, F.S., Tsibris, J.C.M. and Spellacy, W.N. (1983) Contraception 28, 413-422. Beckman, W.C., Hudspeth, D.A., Golding, T., Hubbert, L., Akkerman, J. and Korach, K.S. (1989) Endocrinology 124, 2651-2658. Benoit, V., Valette, A., Mercier, L., Meignen, J.M. and Boyer, J. (1982) Biochem. Biophys. Res. Commun. 109, 11861191. Bjorntorp, P. (1988) Acta Med. Stand. Suppl. 723, 121-134. Bjorntorp, P., Ottosen, M., Rebuff&Strive, M. and Xu, X. (1990) in Obesity: Towards a Molecular Approach (Bray, G.A., Ricquier, D. and Spigelman, B.M., eds.), pp. 147157, Wiley-Liss, New York. Boyd-Leinen, P.A., Fournier, D. and Spelsberg, T.C. (1982) Endocrinology 111, 30-36. Colvard, D.S., Jankus, W.R., Berg, N.J., Graham, M.L., Jiang, N.-S., Ingle, J.N. and Spelsberg, T.C. (1988) Clin. Chem. 34, 363-369. De Pegola, G., Xu, X., Yang, S., Giorgino, R. and Bjorntorp, P. (1991) Int. J. Obesity 15, 54-54. Freedman, D.S., Jacobsen, J.S., Barboriak, J.J., Sobocinski, K.A., Anderson, A.J., Kissebah, A.H., Sasse, E.A. and Gruchow, H.W. (1990) Circulation 81, 1498-1506. Fried, S.K. and Kral, J.G. (1987) Int. J. Obesity 11, 129-140. Gehring, U. and Tomkins, G.M. (1974) Cell 3, 301-306. Gray, J.M. and Wade, G.N. (1979) Endocrinology 104, 13771382. Gray. J.M. and Wade, G.N. (1980) Am. J. Physiol. 239, E237-E241. Gray, J.M., Dudley, S.D. and Wade, G.N. (19811 Am. J. Physiol. 240, E43-E46.
Hietanen, E. and Greenwood, M.C.R. (1977) J. Lipid Res. 18, 480-490. Krotkiewski, M. and Bjorntorp, P. (1978) J. Endocrinol. Invest. 1, 364-371. Krotkiewski, M., Bjorntorp, P., Sjostrom, L. and Smith, U. (1983) J. Clin. Invest. 72, 1150-1162. Leake, R.E., Laing, L., Calman, K.C., MacBeth, F.R., Crawford, D. and Smith, D.C. (1981) Br. J. Cancer 43, 59-65. Lundgreen, H., Bengtsson, C., Blohme, G., Lapidus, L. and SjGstrGm, L. (1989) Int. J. Obesity 13, 413-423. MacFarlane, J.K., Fleiszer, D. and Fazekas, A.G. (1980) Cancer 45, 2998-3003. Munson, P.J. and Rodbard, D. (1980) Anal. Biochem. 107, 220-239. Pasquier, Y.-N., Pecquery, R. and Guidicelli, Y. (1988) Biochem. Biophys. Res. Commun. 154, 1151-1159. Pecquery, R., Leneveu, M.C and Giudicelli, Y. (1986) Endocrinology 118, 2210-2216. Pedersen, O., Hjollund, E. and Lindskov, H.O. (1982) Am. J. Physiol. 243, E158-E167. Pedersen, S.B., Borglum, J.D., Eriksen, E.F. and Richelsen, B. (1991) Biochim. Biophys. Acta 1093, 80-86. Rebuff&Strive, M. (1987a) Acta Physiol. Stand. 129, 471-477. Rebuff&Strive, M. (1987bI Acta Med. Stand. Suppl. 723, 143-146. Rebuff&Strive, M., Enk, L., Crona, N., L&troth, P., Abrahamsson, L., Smith, U. and Bjorntorp, P. (1985) J. Clin. Invest. 75, 1973-1976. Rebuff&Strive, M., Bronnegard, M., Nilsson, A., Eldh, J., Gustafsson, J.-A. and Bjorntorp, P. (1990) J. Clin. Endocrinol. Metab. 71, 1215-1219. Richelsen, B. and Pedersen, 0. (1985) Endocrinology 116, 1182-1187. Richelsen. B., Eriksen, E.F., Beck-Nielsen, H. and Pedersen, 0. (1984) J. Clin. Endocrinol. Metab. 59, 7-12. Ryan, E.A. and Enns, L. (1988) J. Clin. Endocrinol. Metab. 67, 341-347. Shapiro, D.J., Barton, M.C., Mckearin, D.M. et al. (1989) Recent Prog. Horm. Res. 4.5, 29-58. Spelsberg, T.C., Graham, M.K., Berg, N.J., Umehara, T., Riehl, E., Coulam, C.B. and Ingle, J.N. (1987) Endocrinology 121, 631-644. Steingrimsdottir, L., Brasel, J. and Greenwood, M.R.C. (1980) Am. J. Physiol. 239, E162-E167. Stone, G.M. (1963) J. Endocrinol. 27, 281-288. Umehara, T., Graham, M.L., Berg, N.J., Lieber, M.M. and Spelsberg, T.C. (1988) J. Steroid Biochem. 31, 15-25. Vague. J., Meignen, J.M. and Negrin, J.F. (1984) Worm. Metab. Res. 16, 380-381. Wade, G.N. and Gray, J.M. (1978) Endocrinology 103, 16951701.
