ABSTRACT
Leptin is the product of the ob gene, reported to be secreted exclusively from adipocytes and thought to control satiety by providing information to the central nervous system. However, the function of leptin appears to be more complex because multiple studies demonstrate its role in hematopoiesis, reproduction, and immunity. In addition, several nonadipose sources of leptin have been reported. The purpose of this study was to examine several breast cancer cell lines and ductal carcinomas of the breast for expression of leptin messenger RNA (mRNA) and protein. For tumor studies, specimens were preassayed for contaminating adipose tissue. Northern blot analyses demonstrated leptin mRNA in several breast cancer cell lines (MCF-7, T47D, and MDA-MB-231), a normal breast epithelial cell line (MCF10A), and four breast tumors. Leptin protein was identified in T47D breast cancer cells by indirect immunofluorescent staining and in samples of the same breast tumors used for Northern studies by enzyme-linked immunosorbent assays (ELISA). This preliminary study suggests that leptin is expressed in malignant epithelial cells of the breast. Further investigation is needed to determine whether this protein plays a role in breast carcinogenesis.
Subject(s)
Breast Neoplasms/metabolism , Breast/chemistry , Proteins/analysis , Adipose Tissue/chemistry , Breast Neoplasms/genetics , Cell Line , Fluorescent Antibody Technique , Humans , Leptin , RNA, Messenger/analysis , Tumor Cells, CulturedABSTRACT
Sex steroids are postulated to play a role in adipose tissue regulation and distribution, because the amount and location of adipose tissue changes during puberty and menopause. Because of the nature of adipose tissue, receptors for the female sex steroids have been difficult to demonstrate. To date, estrogen receptor messenger RNA and protein have been identified in human subcutaneous adipose tissue, but the presence of progesterone receptor (PR) has not been reported. In this study, we demonstrate PR message by Northern blot analysis in RNA isolated from the abdominal subcutaneous adipose tissue of premenopausal women. These preliminary studies revealed that PR messenger RNA levels are higher in the stromal-vascular fraction as opposed to the adipocyte fraction. Western blot analysis demonstrates both PR protein isoforms (human PR-A and human PR-B) in human subcutaneous adipose tissue. Using an enzyme-linked immunosorbent assay, total PR could be quantitated. These studies substantiate that sex steroid receptors are present in human adipose tissue, thereby providing a direct route for regulation of adipose tissue by female sex steroids.
Subject(s)
Adipose Tissue/chemistry , RNA, Messenger/analysis , Receptors, Progesterone/genetics , Abdomen , Adult , Blotting, Northern , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Female , Humans , Middle Aged , Myometrium/chemistry , PremenopauseABSTRACT
OBJECTIVE: The purpose of this study was to evaluate the regulation of lipoprotein lipase activity, protein mass, and messenger ribonucleic acid by estradiol. STUDY DESIGN: Premenopausal women not taking exogenous sex steroids had transdermal 17 beta-estradiol and placebo patches placed in the gluteal region during the early follicular phase of the menstrual cycle. Adipose biopsies were performed from beneath the patches. Adipose tissue lipoprotein lipase activity was determined by a radiometric assay, protein mass was determined by enzyme-linked immunosorbent assay, and messenger ribonucleic acid level was determined by Northern analysis. Comparisons between the treated and placebo sides were analyzed by nonparametric statistics. RESULTS: Adipose tissue from beneath the 17 beta-estradiol patch had significantly decreased lipoprotein lipase activity and extracellular protein mass than did adipose tissue from beneath the placebo patch. There was no difference in lipoprotein lipase messenger ribonucleic acid levels. CONCLUSION: Estrogen decreases lipoprotein lipase activity by a posttranscriptional modification of protein levels. A hypothesis of sex steroid regulation of body fat distribution is proposed.
Subject(s)
Adipose Tissue/enzymology , Body Composition/physiology , Estradiol/pharmacology , Lipoprotein Lipase/analysis , Adipose Tissue/metabolism , Adipose Tissue/pathology , Administration, Cutaneous , Biopsy , Blotting, Northern , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Estradiol/administration & dosage , Estradiol/analysis , Estrone/analysis , Female , Follicular Phase/physiology , Humans , Lipoprotein Lipase/genetics , Lipoprotein Lipase/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RadiometryABSTRACT
OBJECTIVE: To determine the levels of free estrone (E1) and estradiol (E2) in breast adipocytes of premenopausal women, premenopausal women using oral contraceptives (OCs), postmenopausal women, and postmenopausal women using estrogen replacement therapy (ERT). METHODS: Breast adipose tissue was obtained from 36 premenopausal and 29 postmenopausal women, and adipocytes were separated from stromal and epithelial cells through collagenase digestion and centrifugation. Oil was rendered from adipocytes, and E1 and E2 levels were measured by specific radioimmunoassays after extraction with methanol-water. RESULTS: Estrone and E2 levels were approximately 2.4- and 7.8-fold higher, respectively, in premenopausal women than in postmenopausal women. In premenopausal women, E1 and E2 correlated with the time since last menses (R2 = .55 and .62, respectively), whereas in postmenopausal women, E1 and E2 correlated with body mass index (BMI) (r = .48 and .52, respectively). Estrone levels were always greater than E2 levels in adipocytes, with the E1/E2 ratio being 2.7-fold higher in postmenopausal women than in premenopausal women. The use of OCs decreased E1 and E2 levels in premenopausal women, and ERT increased levels in postmenopausal women. CONCLUSION: Free estrogen in breast adipocytes is characterized by E1 dominance, with levels in premenopausal women correlating with the menstrual cycle and levels in postmenopausal women correlating with BMI.
Subject(s)
Adipocytes/chemistry , Breast/chemistry , Contraceptives, Oral, Hormonal , Estradiol/analysis , Estrogen Replacement Therapy , Estrone/analysis , Postmenopause , Premenopause , Adult , Body Mass Index , Breast/cytology , Case-Control Studies , Female , Humans , Menstrual Cycle , Middle Aged , RadioimmunoassayABSTRACT
OBJECTIVE: To determine the relative amounts of RNA and DNA from adipocytes and adipose stromal-vascular cells to better assess differential gene expression for estrogen receptor and cytochrome P450 aromatase in the two cell fractions of adipose tissue. STUDY DESIGN: Colorimetric and fluorometric assays were used to measure total RNA and DNA, and the relative cell numbers as well as the relative contribution of RNA from adipocytes and stromal-vascular cells were assessed. RESULTS: Adipocytes and stromal-vascular cells exist in a ratio of 2/1. RNA/DNA ratios are the same in the two cell fractions. CONCLUSION: In human subcutaneous adipose tissue, adipocytes are twice as numerous as stromal-vascular cells. The overall transcriptional activity of the cell types appears similar.
Subject(s)
Adipocytes/metabolism , Adipose Tissue/metabolism , Aromatase/genetics , Receptors, Estrogen/genetics , Adipose Tissue/pathology , Adult , Calorimetry , Cell Count , Cytochrome P-450 Enzyme System , DNA/metabolism , Female , Fluorometry , Gene Expression , Humans , Middle Aged , RNA/metabolism , Stromal Cells/metabolismABSTRACT
Clinical evidence suggests that sex hormones affect adipose tissue metabolism and deposition. To investigate the biosynthesis and possible action of estrogen in adipose tissue, we report the use of competitive, specific polymerase chain reaction amplifications to determine levels of estrogen receptor (ER) messenger RNA (mRNA) and cytochrome P450 aromatase mRNA in adipocytes and adipose stromal cells. This extremely sensitive technique uses coamplification of a homologous animal species complementary RNA to control for differences in amplification efficiencies. The DNA amplification products are identified by Southern hybridization with species-specific radiolabeled oligonucleotide probes. Abdominal adipose tissue obtained from female patients during elective abdominoplasty was separated by collagenase digestion and centrifugation into floating adipocytes and pelleted adipose stromal cells. Our results demonstrate higher ER mRNA levels in adipocytes compared to adipose stromal cells, whereas cytochrome P450 aromatase mRNA levels are higher in adipose stromal cells compared to adipocytes. The finding of ER mRNA in adipose tissue suggests the presence of the ER in adipose tissue. In addition the inverse correlation of ER mRNA and cytochrome P450 aromatase mRNA levels in adipocytes and adipose stromal cells suggests a paracrine relationship whereby estrogen produced by adipose stromal cells affects adjacent adipocytes.
Subject(s)
Adipocytes/metabolism , Adipose Tissue/cytology , Aromatase/biosynthesis , Receptors, Estrogen/biosynthesis , Stromal Cells/metabolism , Animals , Aromatase/genetics , Base Sequence , Blotting, Northern , DNA Primers/chemistry , Female , Humans , Mice , Middle Aged , Molecular Sequence Data , Oligonucleotide Probes/chemistry , Oligonucleotides , Polymerase Chain Reaction , RNA, Messenger/analysis , Rats , Receptors, Estrogen/genetics , Transcription, GeneticABSTRACT
We have examined the relationship between total plasma cortisol concentration and DNA repair capacity in human peripheral lymphocytes cultured in vitro; the data indicate that high concentrations of cortisol (> 20 micrograms/dl) inhibit DNA repair. The inhibitory effect can be abrogated by the addition of RU38486, a cortisol antagonist. In addition, we compared plasma cortisol concentration and in vitro DNA repair capacity in 52 healthy individuals. Females on therapeutic estrogen (oral contraceptive or estrogen replacement therapy) had significantly elevated plasma cortisol and suppression of DNA repair capacity.
