Adipose Tissue Stromal Vascular Cells Research Articles

Isolation of Murine Adipose-Derived Stromal/Stem Cells for Adipogenic and Osteogenic Differentiation or Flow Cytometry-Based Analysis.

Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Adipose tissue stromal vascular cells or primary adipocytes derived from murine adipose tissue and grown in culture are essential tools for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stromal/stem cells, along with protocols for inducing adipogenesis to white or beige adipocytes in this cell population and osteogenic differentiation. Isolation of the adipose stromal vascular fraction cells for flow cytometric analysis is also described.

140 Relaxin Regulates Porcine Adipose Tissue Development by Inhibiting Preadipocyte Number, Stimulating Lipolysis, and Upregulating mRNA Expression of Adipokine, Fatty Acid Metabolism and Extracellular Matrix Genes

Abstract Relaxin (RLN) is best known as a reproductive hormone that remodels cervical connective tissue. However, porcine adipose tissue can secrete and respond to RLN, suggesting locally derived RLN could modulate adipose tissue development and function. To test this hypothesis, primary cultures of porcine subcutaneous adipose tissue stromal-vascular (AT-SV) cells and visceral adipose tissue explants were utilized as model systems to determine the effect of 100 ng/ml exogenous RLN on 1) preadipocyte proliferation and differentiation, 2) the mRNA expression of adipokine, extracellular matrix protein and fatty acid metabolism genes, and 3) lipolytic rate in porcine adipose tissue. AT-SV cells were harvested from 3-day-old neonatal pigs (n=6) and plated in vitro in growth medium containing either vehicle or RLN for cell number assays or were induced to differentiate by treatment with insulin, hydrocortisone, and rosiglitazone for 8 days in the presence or absence of RLN for adipogenesis assays. Total RNA was extracted from differentiating cultures on days 0 and 8 post-induction. Real-time PCR was then utilized to determine changes in mRNA expression for target genes. For lipolysis and cell signaling experiments, visceral adipose tissue was obtained from market weight pigs at harvest, minced into explants, and cultured in serum-free medium in the presence or absence of RLN and cAMP pathway modulators with differences in glycerol release determined following 2-hour treatment. Overall, RLN decreased preadipocyte number 1.27-fold under serum-free conditions (P < 0.05), enhanced adipogenesis based upon 2.4-fold increase in GAPDH activity (P < 0.05) and a 2.2-fold increase in ORO accumulation (P < 0.05) while significantly increasing the mRNA expression of multiple metabolic, adipokine, and extracellular matrix component genes. RLN treatment stimulated glycerol release by 3-fold via the cAMP pathway (P < 0.01). Collectively, these data support the hypothesis that RLN regulates adipose tissue development through stimulating adipogenesis and modulating adipocyte metabolism.

14 Characterization of adipose tissue extracellular matrix component mRNA expression during porcine adipogenesis using real-time PCR

Abstract Given adipose tissue is histologically classified as connective tissue, we hypothesized expression of extracellular matrix (ECM) components are significantly altered during adipogenesis. However, little is known about the regulation of the ECM during adipose tissue development in the pig. Therefore, the objective of this study was to characterize expression of ECM components during porcine adipogenesis. Primary cultures of adipose tissue stromal-vascular cells were harvested from 3-day-old neonatal pigs (n=6) and preadipocytes induced to differentiate in vitro for 8 days in the presence of insulin, hydrocortisone, and rosiglitazone. Total RNA was extracted from these cultures on days 0 and 8 post-induction. Real-time PCR was then utilized to determine changes in mRNA expression for collagen type I alpha 1 chain (COL1A), collagen type I alpha 2 chain (COL2A), collagen type I alpha 3 chain (COL3A), collagen type I alpha 4 chain (COL4A), collagen type I alpha 6 chain (COL6A), biglycan, fibronectin, laminin, nitogen-1 (NID1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 9 (MMP9), metallopeptidase inhibitor 3 (TIMP3). The mRNA abundances of COL1A, COL3A and MMP2 were significantly downregulated 2.86-fold (P < 0.05), 16.7-fold (P < 0.01) and 3.1-fold (P < 0.05) respectively in day 8 (differentiated) compared to day 0 (undifferentiated) cultures. Meanwhile, mRNA abundances were significantly upregulated during adipogenesis for the COL2A (2.82-fold; P < 0.05), COL4A (2.01-fold; P < 0.05), COL6A (2.8-fold; P < 0.05), biglycan (49.9- fold; P < 0.001), fibronectin (452-fold; P < 0.001), laminin (6.1-fold; P < 0.05), NID1(47.4-fold; P < 0.01), MMP9 (76.8- fold; P < 0.01), and TIMP3(3.04-fold; P < 0.05) genes. These data support the hypothesis that significant changes in ECM components occur during porcine adipogenesis. Modulating adipose tissue ECM remodeling might be a novel strategy to manipulate adiposity in the pig.

Acute Aerobic Exercise Remodels the Adipose Tissue Progenitor Cell Phenotype in Obese Adults.

Adipose tissue pathology in obese patients often features impaired adipogenesis, angiogenesis, and chronic low-grade inflammation, all of which are regulated in large part by adipose tissue stromal vascular cells [SVC; i.e., non-adipocyte cells within adipose tissue including preadipocytes, endothelial cells (ECs), and immune cells]. Exercise is known to increase subcutaneous adipose tissue lipolysis, but the impact of exercise on SVCs in adipose tissue has not been explored. The purpose of this study was to assess the effects of a session of exercise on preadipocyte, EC, macrophage, and T cell content in human subcutaneous adipose tissue. We collected abdominal subcutaneous adipose tissue samples from 10 obese adults (BMI 33 ± 3 kg/m2, body fat 41 ± 7%) 12 h after a 60 min acute session of endurance exercise (80 ± 3%HRpeak) vs. no acute exercise session. SVCs were isolated by collagenase digestion and stained for flow cytometry. We found that acute exercise reduced preadipocyte content (38 ± 7 vs. 30 ± 13%SVC; p = 0.04). The reduction was driven by a decrease in CD34hi preadipocytes (18 ± 5 vs. 13 ± 6%SVC; p = 0.002), a subset of preadipocytes that generates high lipolytic rate adipocytes ex vivo. Acute exercise did not alter EC content. Acute exercise also did not change total immune cell, macrophage, or T cell content, and future work should assess the effects of exercise on subpopulations of these cells. We conclude that exercise may rapidly regulate the subcutaneous adipose tissue preadipocyte pool in ways that may help attenuate the high lipolytic rates that are commonly found in obesity.

Isolation of Murine Adipose-Derived Stromal/Stem Cells for Adipogenic Differentiation or Flow Cytometry-Based Analysis.

Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Adipose tissue stromal vascular cells or primary adipocytes derived from murine adipose tissue and grown in culture are essential tools for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stromal/stem cells along with protocols for inducing adipogenesis in this cell population or isolating the adipose stromal vascular fraction cells for flow cytometric analysis.

Adipocyte ADRB3 Down-Regulated in Chinese Overweight Individuals Adipocyte ADRB3 in Overweight

Objectives: Activation of β3-adrenoceptor (ADRB3) is essential in the process of human adipose tissue browning, but obese subjects suffered from reduced ability of brown adipose tissue activation. The present study aims to detect the adipocyte ADRB3 expression in overweight individuals and the relationship between adipocyte ADRB3 expression and adiposity in adults. Methods: Visceral adipose tissue samples were obtained from 85 subjects who underwent abdominal surgery. ADRB3 mRNA and protein expression levels in mature adipocytes and adipose tissue stromal vascular cells were examined by quantitative real-time PCR and Western blot assay, respectively. UCP-1mRNA expression levels in mature adipocytes were examined by quantitative real-time PCR. Results: The data revealed that ADRB3 mRNA (p = 0.021) and protein (p = 0.025) expression levels in mature adipocytes were significantly higher in the normal-weight than in the overweight group. Similar results were also found for ADRB3 mRNA (p = 0.041) and protein (p = 0.025) expressions of stromal vascular cells. An inverse correlation was verified between mature adipocyte ADRB3 mRNA expression and BMI (r = –0.362, p = 0.012). UCP-1 mRNA expression levels in mature adipocytes were higher in the normal-weight group compared with the overweight group (p = 0.045). Conclusion: Adipocyte ADRB3 expression levels were down-regulated before the onset of obesity, which indicated that the reduction of ADRB3 expression might be the cause of compromised adipose tissue browning and obesity rather than the result. Thus, the interference of the ADRB3 pathway in adipocytes may provide a potential treatment target for obesity.

Isolation and Analysis of Stromal Vascular Cells from Visceral Adipose Tissue.

The obesity epidemic is the underlying driver of the type 2 diabetes mellitus epidemic. A remarkable accumulation of various pro-inflammatory immune cells in adipose tissues is a hallmark of obesity and leads to pathogenesis of tissue inflammation and insulin resistance. Here, we describe a detailed protocol to isolate adipose tissue stromal vascular cells (SVCs), which enrich various immune cells of adipose tissues. These SVCs can be used to examine the population and activation status of immune cells by tracking their cell surface antigens, gene expression, and activation of specific signaling pathways.

The Control of Stromal Vascular Cell Differetiation by Retinoic Acid and Calcium in Hanwoo Beef Cattle Adipose Tissue

Stromal vascular cells (SVC) from the perirenal adipose tissue of castrated Hanwoo beef cattle steers were isolated and cultured in medium containing different concentrations of calcium and retinoic acid. The cells were stimulated to differentiate in defined medium until lipid accumulation was apparent. The cell viability of adipose tissue SVC were not affected by the different concentrations of calcium and retinoic acid in the media. No observable cytotoxic effect was apparent among the cells treated with various levels of calcium and retinoic acid. Oil-red O staining of differentiated adipose tissue SVC shows that as the level of calcium and retinoic acid decreased, the degree of adipocyte differentiation increased. This shows that manipulating the extracellular level of calcium and retinoic acid in perirenal adipose tissue SVC can result in inhibition or stimulation of adipogenesis. RT-PCR and western blot analysis of adipogenic related genes and proteins clearly showed that the reduction in the level of calcium and retinoic acid in the media, stimulated adipogenesis. The expression level of PPARγ, C/EBPα, SREBP-1c and aP2 were significantly upregulated by the diminution of the calcium and retinoic acid. The expression of major calcium buffering protein calreticulin was also down regulated thus, diminishing its inhibitory effect on PPARγ. Retinoic acid related receptors RARα and CRABP-II were also down regulated upon the reduction of calcium and retinoic acid which may account for the decreased inhibitory effect of retinoic acid on adipocyte differentiation. These results suggest the possible mechanism of action for the increased adipocyte differentiation in Hanwoo beef cattle adipose stromal vascular cells upon decreasing the extracellular calcium and retinoic acid concentration. These findings may be applied in the future to further develop micronutrient manipulations to increase intramuscular fat accumulation in Hanwoo beef cattle.

Characterization of swine adipose tissue monocytic lineage cells

The incidence of obesity is growing in the United States and other developed countries. Macrophage infiltration into adipose tissue is a key characteristic of human obesity. Swine are increasingly used as biomedical models in obesity research due to physiologic similarities of many systems to those of humans. To examine the similarity of monocytic markers in adipose tissue and to further characterize the pig as a biomedical model with which to study obesity, flow cytometry was performed on stromal vascular cells from adipose tissue biopsies of Yorkshire cross pigs. Adipose tissue stromal vascular cells were 34.2% CD172a+ (monocyte) and 26.9% CD203a+(macrophage). The majority of both CD203a+ and CD172a+ cells were CD163+ (69.8% and 59.0%, respectively) and CD14+ (80.5% and 73.3%, respectively). To determine the effect of mild inflammation on adipose tissue macrophage infiltration, swine were injected intravenously with 50 ng/kg body weight lipopolysaccharide (LPS). LPS injection statistically increased the percentage of both CD203+ and CD172a+ cells in adipose tissue at 24 hours after injection. In conclusion, this study utilizes several cell surface markers to characterize the phenotype of monocytic lineage cells in swine adipose tissue and provides valuable data for future studies examining adipose tissue macrophages in obesity.Grant Funding Source : Internal Iowa State University

Lipid and Gene Interactions during Differentiation of Human Subcutaneous Adipose Tissue Stromal Vascular Cells

Examination of the changes in cellular lipid composition, allows for an understanding in their contribution to adipocyte differentiation in health and disease. In the future the analysis of body lipid composition could offer an additional tool to diagnosing disease with the predictive capacity for prognosis. Exposition of cells to excess of energy substrates causes activation of evolutionarily conserved adaptive mechanisms in endoplasmic reticulum stress. In preadipocytes, metabolic stress, through the activation of endoplasmic reticulum stress connected with modification of lipid composition and lipid droplet formation leads to differentiation. The aim of the study was to examine changes in cellular lipid composition in parallel with gene expression and enzyme activity during preadipocyte differentiation using “omics” results. Differentiation condition lead to activation of lipid droplets related protein expression and lipid droplet formation in preadipocytes. The predominant increase of the phospholipids, plasmalogens and cholesterol amounts during cell differentiation was observed. This was accompanied by stimulation of de novo synthesis of saturated fatty acids incorporated into sphingolipids and with reduction in amount of arachidonic acid. Our results indicated that the changes in lipid composition of differentiating stromal vascular fraction cells changed in parallel with gene expression and matched the functional requirements.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: The influence of extracellular matrix on intramuscular and extramuscular adipogenesis1

The extracellular matrix (ECM) and specific ECM components can have a major influence on cell growth, development, and phenotype. The influence of the ECM and ECM components on adipogenesis in vivo and in vitro will be reviewed in this paper. Engelbreth-Holm-Swarm substratum and laminin per se markedly increased attachment, spreading, and hypertrophy of preadipocytes in serum-free primary cultures of porcine adipose tissue stromal-vascular cells. Furthermore, primary cultures of stromal-vascular cells showed that preadipocytes express ECM components after preadipocyte recruitment. Staining for plant lectins, type IV collagen, and laminin in fetal pig adipose tissue demonstrates that adipocyte reactivity for laminin was strong throughout fetal development and was similar for developing adipocytes and vasculature. However, lectin binding and type IV collagen reactivity of blood vessels preceded that for adipocytes. Therefore, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Specific inhibitors and modulators of collagen synthesis have been used to evaluate the role of collagens in the differentiation of bovine intramuscular preadipocytes (BIP) and other preadipocyte cell lines. Triglyceride accretion of BIP cells was inhibited by a general inhibitor of collagen biosynthesis, whereas specific inhibitors or modulators of type IV collagen inhibited 3T3-L1 cell differentiation. Further study revealed that compared with collagens types I to IV, type V and VI collagens have an important and active role in BIP adipogenesis. The growth of intramuscular bovine adipose tissue may be dependent on collagen newly synthesized and organized by the adipocytes per se. The role of extracellular or ECM proteolysis in regulating adipogenesis also will be reviewed in this paper. Many members of the matrix metalloproteinase (MMP) family are expressed by adipocytes, and specific inhibition of MMP-9 greatly reduces adipogenesis in vitro. Possibly, MMP and other proteases regulate turnover of key adipocyte ECM proteins that are involved in the regulation of preadipocyte proliferation and differentiation.

Preadipocyte transplantation: an in vivo study of direct leptin signaling on adipocyte morphogenesis and cell size

Leptin has profound effects on adipose tissue metabolism. However, it remains unclear whether direct leptin signaling in adipocytes is involved. We addressed this question by transplanting inguinal adipose tissue stromal vascular cells (SVCs) from 4- to 5-wk-old wild-type (WT) and leptin receptor-deficient [Lepr(db/db) (db)] mice to inguinal and sternal subcutaneous sites in Ncr nude mice. Both WT and db SVCs gave rise to mature adipocytes with normal morphologies 3 mo after the transplantation. The average adipocyte size (microm(2)/cell) was not significantly different between WT and db transplants at either the inguinal (1,630 +/- 103 vs. 1,491 +/- 74) or the sternal site (1,788 +/- 107 vs. 1,596 +/- 92). Expression levels of beta(3)-adrenergic receptor, a major mediator of lipid mobilization, were indistinguishable between WT and db transplants and similar to those of the hosts. Additionally, adipocyte sizes of inguinal transplants and endogenous inguinal adipose tissues were closely correlated (beta = 0.76, P < 0.001), suggesting that the metabolic milieu of host mice has significant effects on adipocyte size of the transplants. Contrary to the indifference to donor's Lepr genotype, adipocyte size of the transplants was significantly affected by the donor's sex in a leptin receptor-dependent manner. In WT transplants, female SVCs gave rise to smaller adipocytes than male SVCs (1,358 +/- 127 vs. 2,133 +/- 171, P < 0.05). However, this sex difference was not significant in db transplants (1,537 +/- 121 vs. 1,655 +/- 140, P = 0.22). These data suggest that: 1) long-form receptor-mediated direct leptin signaling has no significant cell-autonomous effect on adipocyte differentiation and metabolism in adult mice, 2) sex may affect adipocyte metabolism via genetic and/or epigenetic programming, and 3) leptin may potentiate sexual dimorphism in adipocyte metabolism.

SPARC Inhibits Adipogenesis by Its Enhancement of β-Catenin Signaling

SPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of beta-catenin and subsequently enhanced the interaction of beta-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on beta-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of alpha5 integrin was enhanced, and that of alpha6 integrin was inhibited. Lithium chloride, which enhances the accumulation of beta-catenin, also inhibited the expression of alpha6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation.

CLA differently regulates adipogenesis in stromal vascular cells from porcine subcutaneous adipose and skeletal muscle

Conjugated linoleic acid (CLA), a mixture of isomers of linoleic acid, has previously been shown to be able to decrease porcine subcutaneous (SC) adipose tissue levels while increasing the count of intramuscular (IM) adipose tissue in vivo. However, the underlying mechanisms through which it acts are poorly understood. The objective of this study was to investigate the different effects of CLA on adipogenesis in cultured SC adipose tissue and IM stromal vascular cells obtained from neonatal pigs. As shown here, trans-10, cis-12 CLA decreased the expression of adipocyte-specific genes as well as adipose precursor cell numbers and the accumulation of lipid in cultured SC adipose tissue stromal vascular cells. However, the cis-9, trans-11 CLA did not alter adipogenesis in SC cultures. On the other hand, both CLA isomers increased the expression of adipocyte-specific genes in IM cultures, together with the increasing accumulation of lipid and Oil Red O-stained cells. Collectively, these data show that CLA decreases SC adipose tissue but increases IM adipose tissue by different regulation of adipocyte-specific gene expression. These results suggest that adipogenesis in IM adipocytes differs from that in SC adipocytes.

Modulation of interleukin‐6 (IL‐6) expression and secretion in adipose tissue in vitro and in vivo by n‐3 fatty acids

Obesity is associated increased serum interleukin-6 (IL-6) levels. Adipose tissue has been shown to secret up to 35% of serum IL-6. However, only 10% of adipose tissue-derived serum IL-6 levels are accounted by the fully differentiated adipocytes. Thus, we previously tested and showed that adipose tissue stromal vascular cells and preadipocytes contribute significantly toward obesity-induced increases in plasma IL-6 levels. In our current study, we tested anti-inflammatory effects of n-3 polyunsaturated fatty acids (n-3 PUFA) including eicosapentaenoic acid (EPA, 20:5) and EPA precursor stearidonic acid (SDA, 18:4) on 3T3-L1 preadiopcytes and 3T3-L1 adipocytes. Results showed that EPA (120 μM, 86% decrease) and SDA (120 μM, 82% decrease) significantly reduced lipopolysaccharide (LPS, 200 ng/ml)-induced IL-6 secretion in 3T3-L1 preadiopcytes. Results also showed that EPA (120 μM, 24% decrease) and SDA (120 μM, 39% decrease) significantly reduced LPS-induced IL-6 secretion in 3T3-L1 adipocytes. Further, we showed that n-3 PUFA significantly reduced adipose tissue mesenchymal stem cell IL-6 secretion in a dose dependent manner. These data suggest that n-3 PUFAs have potent anti-inflammatory effects on adipose tissue cell derived IL-6 secretion.

In situ binding and immunocytochemistry of insulin-like growth factor I receptors in primary cultures of porcine adipose tissue stromal vascular cells treated with indomethacin.

An autoradiographic technique for localizing [125I]IGF-I binding was adapted to primary cultures of pig stromal-vascular (S-V) cells to study the effects of pig serum (PS) and (or) indomethacin (Indo) on IGF-I and fibronectin (FN) receptors. Computer assisted image analysis revealed that the percentage of [125I]IGF-I binding decreased (P < .001) in cultures (7 d) treated with Indo compared with 2% PS alone. Binding of [125I]IGF-I analog (low-binding affinity for the IGF-I binding proteins) also decreased (P < .001) in 7-d cultures treated with Indo. The unlabeled IGF-I analog was more effective than unlabeled IGF-I at decreasing (P < .001) binding of [125I]IGF-I. Number of fat cell clusters and fat cell area (total lipid deposition) increased (P < .001) with PS + Indo compared with PS or Indo alone. Immunocytochemical staining (fluorescein isothiocyanate) of IGF-I receptors with a polyclonal antibody (flow cytometric analysis) decreased (P < .001) from 1 to d 3 and did not change from 3 to 7 d with PS alone. However, the percentage of reactive cells decreased (P < .05) when Indo was added to PS. In contrast, the number of cells detected with the FN receptor antibody increased fourfold (P < .01) from 1 to 7 d in cultures with PS alone and decreased (P < .05) when Indo was present. Undifferentiated cells around fat cell clusters were reactive for the IGF-I receptor, but fat cells showed no reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase activity in human skin fibroblasts grown in cell culture

Recent studies in this laboratory have described an unusual kindred in which gynecomastia resulted from abnormally elevated levels of extraglandular aromatase activity. In order to better understand the molecular mechanisms responsible for the abnormal aromatase activity in these and other patients, we explored the aromatase activity of genital skin fibroblasts. Our studies demonstrate that the kinetic parameters for aromatase in skin are similar to those of other cultured cells and suggest that skin is an important site of extraglandular aromatase activity. These cells also contain 5 alpha-reductase activity and androgen receptors and are, therefore, a model for androgen action and metabolism. For example, they provided a system for the study of the potency and specificity of the aromatase inhibitors 4-OHA and MDL 18,962. Finally, the influence of DEX on aromatase in genital skin fibroblasts differs in some important respects from the pattern of control observed in adipose tissue stromal-vascular cells. These findings suggest that investigating the molecular mechanisms for the regulation of aromatase in skin may provide unique information about the control of the enzyme.

Time-dependent biphasic response of aromatase to dexamethasone in cultured human skin fibroblasts.

Human genital skin fibroblasts grown in cell culture possess aromatase activity and, therefore, provide a model to investigate the molecular mechanisms that control aromatase in extraglandular tissues. Following the observation by other investigators that glucocorticoids stimulated aromatase activity in cultured stromal-vascular cells from adipose tissue, we examined the influence of dexamethasone (DEX) on aromatase in cultured skin fibroblasts. Preincubation of skin fibroblasts with DEX stimulated aromatase expression in all cell strains. In time-course studies, aromatase activity showed a biphasic curve, with peak levels at 12 h and a return to baseline levels by 72 h. When DEX was removed after 12 h, aromatase activity could be completely restimulated by DEX only after a period of 60-72 h. The DEX stimulation appeared to involve glucocorticoid receptor function, since the concentration of DEX required for half-maximal stimulation of aromatase activity (4.2 nM) was similar to the dissociation constant (Kd, 4.3 nM) of the receptor (for DEX). Actinomycin D and cycloheximide (CHX) inhibited DEX stimulation of aromatase when they were present in the preincubation and assay media. When cells were preincubated with DEX and CHX and then washed free of CHX and DEX before the assay, superinduction of aromatase activity occurred. Our data concerning the time course and superinduction of aromatase activity by DEX are in contrast to the findings reported by others for adipose tissue stromal-vascular cells and suggest that the mechanisms for the control of aromatase in extraglandular tissue may vary significantly in different tissues.

Adipocyte development in primary rat cell cultures, effect of cell density and serum source.

The development of adipocytes was studied in primary cultures of rat adipose tissue stromal-vascular cells on collagen-coated glass coverslips. The effects of cell density and serum source on lipoprotein lipase (LPL), esterase and lipid histochemistry were evaluated. With a mixture of fetal calf serum (FCS; 2%), horse serum (2%) and pig serum (PS; 10%), large and loosely arranged clusters of adipocytes developed with time through an increase in cell number and size. An inverse relationship was observed between cell size and the number of adipocytes in a cluster. Lower cell densities were associated with large cells and the densest areas contained smaller cells. Unilocular adipocytes were observed by d 13 after plating and were generally absent from the densest area of the coverslips. Histochemically detectable LPL activity was demonstrable before lipid deposition in adipocyte clusters. A comparison of FCS (10%) and PS (10%) as the only serum sources indicated higher level of adipocyte esterase activity and lipid deposition in PS cultures. Cultures of cells from weanling (21 to 28 d old) and old (18 mo) rats were similar, whereas cells from younger rats (2 to 4 d old) formed denser cultures that contained fewer adipocytes. These adipocytes were small (less than 30 micron) and morphologically homogeneous (all multilocular). When cells from the very young rats (2 to 4 d old) were plated at low densities, an inverse relationship between cell size and number of cells in a cluster was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase activity in cultured human genital skin fibroblasts.

Aromatase activity of human genital skin fibroblasts grown in cell culture was studied using both [1,2,6,7-3H] androstenedione (A) and [1-3H]A as substrates. With the former substrate the generation of [3H]estrogens was determined, whereas with the latter substrate, the formation of [3H]H2O was measured. Our results showed that the release of [3H]H2O from [1-3H]A provides an accurate and sensitive method for determining aromatase activity in cultured human skin fibroblasts. Because genital skin fibroblasts also possess marked 5 alpha-reductase activity, we found that addition of an alternate substrate for 5 alpha-reductase was necessary to prevent shunting of A from the aromatase pathway. Hence, all aromatase assays were carried out in the presence of 5 microM progesterone. Under these experimental conditions, no correlation was found between levels of 5 alpha-reductase and aromatase activities. The Michaelis-Menten constant (Km) of the aromatase in cultured genital skin fibroblasts measured in the presence of A and added progesterone ranged between 10 and 39 nM, and the maximum velocity (Vmax) ranged between 0.14 and 1.46 pmol product/mg protein/h. These values are in good agreement with those previously described for adipose tissue stromal-vascular cells, suggesting that the aromatase complexes are similar in skin and adipose tissue. We conclude that skin may be an important site for aromatization of androgens to estrogens in men.