Biochemical and immunological characterization of oestrogen receptor in the cytosolic fraction of gluteal, omental and perirenal adipose tissues from sheep

Abstract

Determination of the presence and characterization of oestrogen receptors (ERs) in subcutaneous and internal fat depots were performed and compared with ERs in the uterus using ligand binding and immunological techniques. Successful and consistent measurement of ERs in ovine adipose tissue could only be accomplished in animals depleted of endogenous sex steroids by combined ovariectomy and adrenalectomy. Scatchard, sucrose gradient and Western blot analyses all confirmed the presence of ERs in the cytosolic fractions of various adipose and uterine tissues from ovariectomized-adrenalectomized ewes. The approximate Kd values of 0.1-0.4 nmol/l for oestradiol binding in cytosolic fractions of gluteal, omental and perirenal adipose tissues were similar to the expected high affinity binding of Kd 0.35 nmol/l observed in uterine tissue. The binding was specific for oestrogens, as unlabelled diethylstilboestrol and oestradiol effectively competed with labelled hormone for receptor sites and progesterone, R5020, testosterone and dexamethasone all failed to compete. Mean (+/- S.E.M.) concentrations of ERs, expressed as fmol specific binding sites per mg protein, were much lower (P < 0.05) in adipose tissues than in uterine tissue (975 +/- 33). However, the content of ERs was greater (P < 0.05) in subcutaneous gluteal fat (11.5 +/- 0.8) than in the internal omental or perirenal fat (5 +/- 0.6) depots. ERs from adipose and uterine tissues both migrated as moieties of 8S on 5-20% sucrose gradients. Western blot analysis of ERs from uterine and adipose tissues in the presence of protease inhibitors demonstrated an immunostaining band with a molecular mass of 67 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Progesterone Receptor in Human Subcutaneous Adipose Tissue

“…In past studies, ERs could not be demonstrated with routine binding assays because of the high nonspecific binding of estrogens stored in adipose tissue (14). With the development of more sensitive assays, ER mRNA (by PCR and Northern blot analysis) and ER protein (8,9) have been demonstrated in human adipose tissue.…”

Pathophysiology of Human Visceral Obesity: An Update

“…Direct action of estrogens in adipose tissue is supported by the presence of both receptor isoforms ␣ and ␤ (94, 124). Sex-and depotrelated differences in estrogen receptor levels have been reported, but the data remain unclear (54,124,421,609). Interestingly, deletion of the estrogen receptor ␣ in male and female mice is associated with increased adiposity independent of food intake (216).…”

Subcellular distribution and glycosylation pattern of androgen receptor from sheep omental adipose tissue

“…Oestrogen receptor (ER) was present in adipose tissues from these animals (Watson et al 1993) and the ER appeared to be functional as oestrogen treatment of ovariectomized ewes resulted in the up-regulation of progesterone receptor (PR) in adipose tissues (Mayes et al 1996). This study is our initial investigation into the presence of AR in omental adipose tissue from female sheep.…”

“…Omental adipose tissue (1 g/ml) was homogenized at 4 C with Medium I (0·25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7·4) which contained protease inhibitors (aminoethyl-benzenesulfonyl fluoride, leupeptin, bacitracin, aprotinin and pepstatin; Watson et al 1993). Unless otherwise stated, all chemicals and reagents were obtained from Sigma (St Louis, MO, USA).…”