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Adipocyte Differentiation

ABSTRACT: Fat tissue was resected during gastric bypass surgery for management of obesity. All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin. Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37*C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer. Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 *M rosiglitazone, antibiotics, and 540 *M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days. For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol. RNA was isolated from preadipocytes by the Trizol method. RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis. Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm. Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings. Keywords = adipocyte Keywords = subcutaneous Keywords = mesenteric Keywords = omental Keywords: repeat sample

ORGANISM(S): Homo sapiens

PROVIDER: GSE1657 | GEO | 2005/06/01

SECONDARY ACCESSION(S): PRJNA90679

REPOSITORIES: GEO

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Project description:Fat tissue was resected during gastric bypass surgery for management of obesity. All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank?s balanced salt solution with bicarbonate, penicillin, and gentamicin. Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37*C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer. Cells were plated in 1:1 Dulbecco?s modified Eagle?s medium:Ham?s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 *M rosiglitazone, antibiotics, and 540 *M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days. For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol. RNA was isolated from preadipocytes by the Trizol method. RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis. Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm. Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.

Project description:Full title: Expression data from human primary subcutaneous preadipocytes treated with glucocorticoids prior to the initiation of differentiation. Preadipocytes are continuously exposed to glucocorticoids in situ due to both steroid present in the circulatory system as well as adipose tissue specific 11Î²HSD1 activity. While the effects of glucocorticoids during differentiation are well studied, the effect of exposure of preadipocytes to glucocorticoids prior to differentiation is unknown. We therefore treated confluent human primary preadipocytes drived from subcutaneous adipose tissue with the synthetic glucocorticoid dexamethasone for 48 hours prior to the initiation of differentiation and assessed what effect this had on their subsequent potential to differentiate. We found that pretreatment with glucocorticoids had a priming effect and resulted in increased differentiation of these preadipocytes. Furthermore, this treatment was additive to the effects of glucocorticoids during the initial phase of adipogenesis. Microarray analysis performed subsequent to the pretreatment with glucocorticoids (at the time point at which preadipocytes would have been induced to differentiate) identified glucocorticoid-responsive, candidate genes whose altered expression could mediate these effects. keywords: glucocorticoids, glucocorticoid receptor, preadipocytes, adipogenesis, human primary preadipocytes, subcutaneous, adipose tissue Experiment Overall Design: Human subcutaneous primary preadipocytes were purchased from Zen-Bio Inc. Preadipocytes from 5 female donors (average BMI 22.5Â±0.2kg/m2) were pooled prior to the initial seeding in T75 flasks. Cells were maintained at 5% CO2 in DMEM with 1.0g/L glucose, 20% calf serum, 100U/ml penicillin, 100mg/ml streptomycin and 50U/ml nystatin. Cells were expanded once prior to seeding in Nunc-brand 12-well dishes. Upon reaching confluence (24 h post-splitting), preadipocytes were stimulated with vehicle or 1uM dex for 48 hours in growth media containing 3% calf serum. Microarray analysis was performed on duplicate samples.

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Adipocyte Differentiation

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