Stromal-vascular Research Articles

Monocyte chemoattractant protein-1 is produced in isolated adipocytes, associated with adiposity and reduced after weight loss in morbid obese subjects

Obesity is associated with insulin resistance and premature atherosclerosis. The human adipose tissue produce several adipokines including monocyte chemoattractant protein (MCP)-1, associated with cardiovascular disease and found to be involved in the pathogenesis of atherosclerosis in vitro. (1) To compare mRNA levels of MCP-1, leptin and a macrophage-specific marker (CD68) in isolated adipocytes vs stromal-vascular (SV) cells, (2) to compare mRNA levels of MCP-1 in human adipose tissue to circulating MCP-1 and adiposity (eg BMI: kg/m2) and (3) investigate the effect of weight loss in obese subjects on circulating MCP-1 and leptin. (1) MCP-1 and CD68 mRNA levels in isolated adipocytes vs SV cells were 17% (P<0.01) and approximately 2% (P<0.001), respectively. Leptin mRNA levels in SV cells were approximately 1% of that in isolated adipocytes (P<0.01). (2) MCP-1 mRNA levels correlated with circulating MCP-1 (P<0.05) and BMI (P<0.05). (3) A 12% weight loss (P<0.001) was associated with a 25% decrease in insulin levels (P<0.01). Circulating MCP-1 and leptin decreased by 20% (P<0.001) and by 24% (P<0.001), respectively. The findings demonstrate that MCP-1 is produced in isolated human adipocytes. In addition, the findings suggest that MCP-1 may be involved in obesity-related health complications and support the hypothesis that weight loss is beneficial by improving the low-grade inflammation observed in obesity.

Recruitment and differentiation of intramuscular preadipocytes in stromal-vascular cell cultures derived from neonatal pig semitendinosus muscles.

The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte recruitment and expression of CCAAT/enhancing binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) proteins in stromal-vascular (SV) cell cultures derived from neonatal subcutaneous adipose tissue and semitendinosus muscles. One adipose tissue SV cell culture and one semitendinosus muscle SV cell culture were established from each of six young pigs (5 to 7 d of age). Conventional SV cell-culture procedures were used to digest adipose and muscle tissue and to harvest and culture adipose and muscle SV cells. Muscles were digested after the removal of all visible connective tissue from the excised muscle. One hour after seeding, muscle SV cell cultures were rinsed and refed new media to remove debris and insoluble muscle protein. The SV cell cultures were double-stained for lipid and the AD-3 antibody, a preadipocyte marker, at 1, 3, and 6 d and were double-stained for lipid and C/EBPalpha or PPARgamma at d 6. Preadipocytes were randomly distributed and not clustered after 1 d in muscle and adipose SV cultures. Regardless of treatment, relative and absolute fat cell numbers were lower (P < 0.05) in muscle than in adipose-SV cell cultures. The DEX treatments produced similar magnitudes of increase in relative and absolute preadipocytes and adipocytes in muscle- and adipose-SV cultures. Several extracellular matrix substrata had no influence on adipogenesis in muscle-SV cell cultures. These studies indicate that muscle-SV cultures are characterized by a low number of adipocytes under basal conditions and a low number of glucocorticoid-responsive preadipocytes.

Dexamethasone induced preadipocyte recruitment and expression of CCAAT/enhancing binding protein α and peroxisome proliferator activated receptor-γ proteins in porcine stromal-vascular (S-V) cell cultures obtained before and after the onset of fetal adipogenesis

The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte recruitment and expression of transcription factor proteins in adipose tissue stromal-vascular (S-V) cell cultures from 50 and 75 day old pig fetuses and young pigs. C/EBPα, C/EBPδ, and PPARγ immunoreactive cells had evenly reactive nuclei and unreactive nucleoli. DEX recruited many more preadipocytes in 75 day than in 50 day fetal S-V cultures. However, DEX did not increase the number of differentiated preadipocytes (lipid+, C/EBPα+) in 50 day S-V cultures and only slightly increased this number in 75 day fetal S-V cultures. In fetal cultures, extensive, precocious increases in C/EBPα expression (number of reactive cells) by day three were followed by extensive decreases in expression. However, PPARγ expression was not expressed precociously since preadipocyte lipid accretion and PPARγ immunoreactivity were strongly linked in fetal and pig S-V cultures. Nevertheless, all cells with lipid in fetal S-V cultures were C/EBPα and PPARγ reactive. DEX increases preadipocyte differentiation in pig S-V cultures and in this study DEX increased PPARγ expression to a much greater degree in pig than in fetal S-V cultures. These studies suggest that restricted adipogenesis in the pig fetus is attributable to limited DEX induced PPARγ expression.

Angiogenesis in ductal carcinoma in situ (DCIS) of the breast

Background Up to 50% of recurrences of ductal carcinoma in situ (DCIS) of the breast are associated with invasive carcinoma but no pathological or molecular features have yet been found to predict for the development of invasive disease. For a tumour to invade, it requires the formation of new blood vessels. Previous studies have described a vascular rim around ducts involved by DCIS raising the possibility that the characteristics of periductal vascularisation may be important in determining transformation from in situ to invasive disease as well as risk of recurrence. Hypothesis I hypothesized that the risk of malignant tranformation (from normal breast to DCIS) and of invasive recurrence, following initial diagnosis and treatment of DCIS, is associated with the extent and pattern of periductal vascularity. It is likely that the periductal vessels are most important in this respect as incipient invasion is most likely to be associated with changes in vessels in the immediate vicinity of the tumour cells. Methodology I investigated to see whether changes in vasculature are related to the progression of in situ to invasive carcinoma and if so, which factors may be important in this change. I initially studied vascular density surrounding DCIS with and without invasive cancer and its relationship to specific angiogenic factors secreted by the tumour. Periductal vascular density and phenotype were determined using morphometry and a panel of anti-endothelial antibodies (von Willebrand factor [vWF], CDS31, CD141 and CDS34). These were related to the presence of invasive carcinoma at initial diagnosis, the histological features of DCIS and the risk of recurrence. For each histological section, the number of stained vessels and endothelial cells within 100 micrometers of foci of DCIS were counted. Up to 50 foci of DCIS on a single slide were scored and the microvessel density (MVD) and endothelial cell density (ED) were calculated for each focus. Normal lobules at least 2 mm away were used as controls. These studies suggested a change in phenotype and dual fluorescence immunostaining for CDS4+ and vWF+ vessels was carried out to confirm the change. The relationship between Thymidine Phophorylase (TP) expression on sections of pure DCIS and periductal vascularity was assessed as it had been previously shown to correlate with stromal vascular density. The relationship between stromal (hotspots) and periductal vascularity was also evaluated. Results Compared to normal lobules, pure DCIS exhibited a greater density of CD34+ and CDS1+ vessels but a decrease in those that were immunopositive for von Willebrand factor (vWF). DCIS associated with invasive carcinoma showed a profile of vascular immunostaining similar to that of pure DCIS but there were statistically significantly greater numbers of CDS4+ and CD141+ vessels and fewer staining for vWF. There was a significant negative correlation between vascular density and both the cross-sectional areas of the ducts involved and the extent of the necrosis of the tumour they contained. A correlation between vascular density and nuclear grade was also noted, being highest in the intermediate grade. On dual staining, the number of vessels stained only by CD34 was significantly higher around DCIS compared to adjacent normal lobules. For TP, although a relationship was seen between the H-score and MVD, a relationship with recurrence was not identified. Vascularity stained by anti-CD34 around DCIS which recurred was statistically significantly higher than those which did not. There was a significant and positive correlation between the two types of vascularity (p<0.001) in sections stained with anti-vWF antibody, but not anti-CD34. Conclusions Blood vessels surrounding DCIS appear to have a different immunophenotype when compared with blood vessels surrounding normal breast lobules. Increases in vascular density, as detected with the CD34 antibody, correlates with recurrence and the development of invasive carcinoma. Recurrent disease does not appear to be related to TP expression by DCIS. Periductal MVD appears to be more important than stromal MVD in predicting for recurrence in DCIS.

The expression of cyclin D1 during adipogenesis in pig primary stromal-vascular cultures.

Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal-vascular (S-V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Western blot analysis was performed on cultured S-V cells using 8% sodium dodecyl sulfate-polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti-mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti-CCAAT/enhancer binding protein (C/EBP alpha) and anti-cyclin D1 together. Cyclin D1 expression was evaluated in 105-day fetal dorsal subcutaneous tissues using paraffin sections. Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S-V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBP alpha beginning at day 3 and remained colocalized with C/EBP alpha through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. The elevated level of cyclin D1 expression colocalized with C/EBP alpha expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid-filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.

Differentiation of stromal-vascular cells isolated from canine adipose tissues in primary culture.

A culture condition supporting adipocyte differentiation of stromal-vascular (S-V) cells isolated from canine adipose tissues was established. Morphological observation and determination of glycerol-3-phosphate dehydrogenase (GPDH) activity were used as the criteria for adipocyte differentiation. After reaching confluence, the cells were able to undergo terminal adipocyte differentiation by treatment with 100 microM indomethacin, 10 microg/ml insulin and 0.5 mM 1-methyl-3-isobutylxanthine (MIX) in medium supplemented with 5% fetal calf serum (FCS). In the absence of either indomethacin or insulin, the S-V cells did not undergo adipose conversion and GPDH activity was not increased, indicating that both indomethacin and insulin play essential roles in this culture system. The S-V cells from inguinal adipose tissues exhibited the greatest increase in GPDH activity among the four depots (inguinal > abdominal-subcutaneous > perirenal > omental). demonstrating that adipocyte differentiation was also intensely dependent on anatomic sites from which the S-V cells were derived. Interestingly, dimethylsulfoxide (DMSO) was found to accelerate adipocyte differentiation in combination with indomethacin and insulin. Under this condition, up to 90% of the cells displayed adipocyte phenotypes and the GPDH activity reached 1288 +/- 441 mU/mg protein. This culture system may be useful for investigating other adipogenic factors as well as anti-adipogenic factors involved in the regulation of canine adipose tissue development.

Inhibition of Chicken Adipocyte Differentiation by in Vitro Exposure to Monoclonal Antibodies Against Embryonic Chicken Adipocyte Plasma Membranes

Specific monoclonal antibodies (MAb) against adipocyte precursor antigens were developed. These MAb identified adipocyte precursors and reduced their prominence in primary stromal-vascular (SV) cultures by complement-mediated cytotoxicity or by inhibition of differentiation. Binding of antibodies to chicken adipocyte precursors was confirmed by immunofluorescence visual examination following secondary exposure to fluorescein isothiocyanate-conjugated goat antimouse IgG. Cross-reaction of MAb with muscle, kidney, liver, fibroblasts, and other cell types not containing lipid droplets was not observed in primary cultures. Adipocyte precursors were obtained from 18-d chick embryo adipose tissue by collagenase digestion to investigate complement-mediated cytotoxicity of preadipocytes. Cultures were maintained in Medium 199 with 5% fetal bovine serum (FBS) for 4 d. Subsequently, Medium 199 supplemented with 10% chicken serum initiated adipocyte differentiation. At Day 5 postinoculation, individual or combinations of MAb were administered to preadipocyte cultures; rabbit complement was added 30 min later. After 1 d of incubation, four of the six individual MAb with complement significantly (P < 0.05) reduced the number of fat cell clusters that developed by 40 to 60%. These MAb in the presence of complement also significantly (P < 0.05) reduced mean cell width and apparent cell area or cell cluster area of lipid-containing cells. Neither MAb nor complement alone reduced fat cell cluster number, cell size, or cluster size. Treatment with pools of two and four MAb decreased the total amount of MAb protein required to reduce fat cell cluster number. Four antibodies, alone or in combination, reduced fat cell cluster development in a complement-dependent manner.

The expression of peroxisome proliferator-activated receptor gamma in pig fetal tissue and primary stromal-vascular cultures.

This study was designed to determine when peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in developing fetal adipose tissue and stromal-vascular adipose precursor cells derived from adipose tissue. In addition we examined developing tissue for CCAAT/enhancer-binding protein beta (C/EBPbeta) expression to see if it was correlated with PPARgamma expression. Pituitary function and hormones involved with differentiation (dexamethasone and retinoic acid) were also tested for their effects on PPARgamma expression to determine if hormones known to affect differentiation also effect PPARgamma expression in vivo and in cell culture. Developing subcutaneous adipose tissues from the dorsal region of the fetal pig were collected at different gestation times and assayed using Western blot analysis to determine levels of PPARgamma and C/EBPbeta. Hypophysectomy was performed on 75-day pig fetuses and tissue samples were then taken at 105 days for Western blot analysis. Adipose tissue was also taken from postnatal pigs to isolate stromal-vascular (S-V) cells. These adipose precursor cells were grown in culture and samples were taken for Western blot analysis to determine expression levels of PPARgamma. Our results indicate that PPARgamma is expressed as early as 50 days of fetal development in adipose tissue and continues through 105 days. Expression of PPARgamma was found to be significantly enhanced in adipose tissue from hypophysectomized fetuses at 105 days of fetal development (p<0.05). C/EBPbeta was not found in 50- or 75-day fetal tissues and was found only at low levels in 105-day tissues. C/EBPbeta was not found in hypophysectomized (hypoxed) 105-day tissue where PPARgamma was elevated. S-V cells freshly isolated from adipose tissue of 5- to 7-day postnatal pigs showed the expression of PPARgamma1. When S-V cells were cultured, both PPARgamma1 and 2 were expressed after the first day and continued as cells differentiated. High concentrations of retinoic acid decreased PPARgamma expression in early S-V cultures (p<0.05). Our data indicate that PPARgamma is expressed in fetal adipose tissue very early before distinct fat cells are observed and can be expressed without the expression of C/EBPbeta. The increase in PPARgamma expression after hypophysectomy may explain the increase in fat cell size under these conditions. Adipose precursor cells (S-V cells) from 5- to 7-day postnatal pigs also express PPARgamma in the tissue before being induced to differentiate in culture. Thus S-V cells from newborn pig adipose tissue are probably more advanced in development than the 3T3-L1 cell model. S-V cells may be in a state where PPARgamma and C/EBPalpha are expressed but new signals or vascularization are needed before cells are fully committed and lipid filling begins.

The influence of dexamethasone and insulin on expression of CCAAT/enhancer binding protein isoforms during preadipocyte differentiation in porcine stromal-vascular cell cultures: evidence for very early expression of C/EBPalpha.

Expression of CAAT/enhancer binding protein (C/EBP) isoforms was examined in primary cultures of adipose tissue stromal vascular (S-V) cells before and during preadipocyte differentiation. Immunocytochemistry showed that the proportions and numbers of C/EBPalpha-, C/EBPbeta-, and C/EBPdelta-reactive cells were maximized after seeding and plating from d 0 to 3 in fetal bovine serum (FBS). However, there were few preadipocytes (AD-3+) and fewer cells with lipid and the number of C/EBPalpha-reactive cells clearly exceeded the number of preadipocytes. Seeding and plating in dexamethasone (DEX) + FBS from d 0 to 3 markedly increased the proportions and numbers of preadipocytes and C/EBPalpha-reactive cells compared to seeding and plating in FBS, d 0 to 3. The number of C/EBPalpha- and C/EBPbeta-reactive cells and preadipocyte reactivity for C/EBPbeta decreased with insulin or DEX treatment, d 3 to 6, following FBS, d 0 to 3. However, insulin + DEX treatment, d 3 to 6, maintained the number of C/EBPalpha-reactive cells and either maintained or increased preadipocyte reactivity for C/EBPalpha and C/EBPbeta. DEX and DEX + insulin treatment induced recruitment of a similar number of preadipocytes, but preadipocytes were not reactive for C/EBPalpha and C/EBPbeta in DEX-treated cultures. The number of C/EBPdelta reactive cells did not change from d 3 to 6 and was not influenced by hormone treatment. After DEX + FBS, d 0 to 3, the high numbers of C/EBPalpha-reactive cells and preadipocytes were maintained by insulin treatment alone. Western blot analysis for C/EBPalpha confirmed the immunocytochemical results. Double staining demonstrated that expression of C/EBPalpha protein was maximized before or at the onset of lipid accretion, whereas expression of C/EBPbeta protein was correlated with lipid accretion. These results indicate that coupling or integration of preadipocyte recruitment with C/EBPalpha expression may be a critical step in glucocorticoid-induced adipogenesis.

Expression of leptin mRNA and CCAAT-enhancer binding proteins in response to insulin deprivation during preadipocyte differentiation in primary cultures of porcine stromal-vascular cells

The aim of this study was to examine the correlation between CCAAT-enhancer binding proteins (C/EBPs) and leptin gene expression in response to insulin deprivation in preadipocytes and adipocytes. Adipose tissue from 7 d-old pigs was digested enzymatically and stromal-vascular (S-V) cells were seeded and plated for 3 d in fetal bovine serum (FBS) with dexamethasone (DEX) followed by 6 d (Days 3–9) in serum-free medium with insulin (850 nM or 10 nM), transferrin, and selenium. During FBS+DEX treatment (Days 0–3) a large number of preadipocytes develop with no lipid accretion. In contrast, preadipocyte number does not change with lipid accretion during insulin treatment (Days 3–9). Total RNA and cells were harvested from S-V cultures after periods with and without insulin after FBS+DEX. Northern-blotting and Western blot analysis were used to study leptin mRNA and C/EBP protein expression in cultures, respectively. Insulin deprivation from Days 3–4 reduced leptin mRNA and C/EBP-α protein expression. Treatment with 850 nM or 10 nM insulin from Days 3–9 induced leptin mRNA and C/EBP-α expression at a similar level. In cultures treated with 10 nM insulin from Days 3–7, leptin and C/EBP-α expression were reduced markedly by insulin deprivation from Days 7–9, but were restored by insulin treatment for 6 hr before harvesting. The restoration of leptin expression by insulin was blocked by cycloheximide treatment. However, C/EBP-β protein levels did not change regardless of insulin deprivation. Insulin deprivation from Days 7–9 in cultures treated with 850 nM insulin from Days 3–7 did not influence C/EBP-α or leptin mRNA expression, whereas C/EBP-α and leptin expression were reduced after treating these cultures with 1.5 uM okadaic acid for 45 min before harvesting on Day 9. However, cycloheximide treatment for 6 hr before harvesting did not reduce leptin mRNA expression. These results suggest that 1) leptin expression is positively correlated with C/EBP-α expression, and 2) the maintenance of leptin expression after insulin deprivation in 850 nM insulin-treated cultures on Day 9 may be associated with the presence of C/EBP-α expression and/or activation.

Adipocyte development is dependent upon stem cell recruitment and proliferation of preadipocytes.

The ability to acquire fat cells persists over the life spans of animals. It is unknown whether adipocyte acquisition is the result of preadipocyte proliferation or stem cell recruitment to become adipocytes. The purposes of these studies were 1) to characterize early differentiation of stromal vascular (S-V) cells to preadipocytes as it is influenced by insulin, dexamethasone (DEX), and insulin-like growth factor-I (IGF-I); and 2) to determine whether new fat cells arise from stem cell recruitment or preadipocyte proliferation. Freshly isolated S-V cells from rat inguinal adipose tissues were plated for 24 hours then exposed to serum-free medium. Approximately 15% of freshly plated S-V cells were preadipocytes as determined by a preadipocyte specific marker, AD3. Total cell number and proportion of preadipocytes were significantly greater with 100 nM insulin treatment than with 0, 0.1, or 1.0 nM, but IGF-I treatment at 10 nM resulted in preadipocyte development similar to that with 100 nM insulin treatment. The addition of 5 nM DEX to the 100 nM insulin treatment resulted in a 20% increase in preadipocyte number by day 2 when compared to either treatment alone. 5-Bromo-2'-deoxy-uridine treatment suppressed the increased proportion of preadipocytes from days 0-2 in non-insulin treated cells and prevented the increase typically observed with insulin. A mitosis inhibitor also significantly reduced the proportion of preadipocytes. These results show for the first time that S-V cells are recruited as preadipocytes and that proliferation of these preadipocytes and early differentiation occur simultaneously.

Expression of CCAAT/enhancer binding protein C/EBPα, β and δ in rat adipose stromal-vascular cells in vitro

Stromal-vascular (S-V) cells from rat inguinal fat depots were isolated and cultured in medium containing fetal bovine serum (FBS) and differentiated in defined medium until lipid accumulation was apparent. C/EBPα, β and δ levels were evaluated for different growth conditions and at different times using Western blots. Immediately after isolation C/EBPα, β and δ could not be detected in S-V cells. After seeding for 24 h in Dulbecco’s modified Eagle’s medium (DMEM) with FBS, C/EBPα, β and δ could all be detected. Cells at day 1 of culture in insulin, transferrin, triiodothyronine and selenium (ITTS) had increased levels of C/EBPα and continued steady high levels to day 6 of culture. Cultures grown in DMEM alone, with no ITTS, showed C/EBPα levels similar to ITTS cultures at day 1 and day 3; however, levels diminished after day 3. DMEM cultures also showed lipid accumulation at day 6; however, the number of cells and the amount of lipid cell were reduced from levels observed in ITTS cultures. C/EBPβ was expressed uniformly throughout the culture period in either DMEM or ITTS cultures while C/EBPδ expression was higher with DMEM treatment than with ITTS. Treatment of 2 day DMEM cultures with FBS increased levels of C/EBPβ and δ but significantly reduced levels of C/EBPα. Immunocytochemical analysis of S-V cells at day 1 of culture showed a similar percentage of cells stained in DMEM cultures and ITTS cultures. However, by day 6 of culture the percentage of cells staining positively for C/EBPα in DMEM had been reduced by one half while in ITTS the percent positive cells remained about the same. Our results indicate that ITTS is not necessary for the induction of C/EBPα and accumulation of lipid in S-V cells. However, ITTS is responsible for maintaining C/EBPα and enhanced lipid accumulation. Because C/EBPα, β and δ expression occurs very early in cell culture and C/EBPα and δ expression continues to increase in DMEM without any apparent inducing agents, our results suggest that these factors may be expressed by the same cells in vivo before being placed in culture. Thus, a large fraction of S-V cells may be further along in the differentiation program than 3T3 cells are when they begin differentiation.

Dehydroepiandrosterone alters the growth of stromal vascular cells from human adipose tissue.

The purpose of this study was to determine if the antiobesity actions of dehydroepiandrosterone (DHEA) observed in vivo are due to an influence on the proliferation and differentiation of primary cultures of stromal-vascular (SV) cells isolated from human adipose tissue. SV cells were isolated from subcutaneous adipose tissue obtained from a young adult female undergoing elective liposuction. For the proliferation assay (Experiment 1), cultures were fed proliferation media containing 0, 5, 25 or 100 microM DHEA for 3d. At the end of this treatment period, cultures were either prepared for counting or for determining their metabolic activity using the Alamar Blue staining procedure. For the differentiation assays (Experiment 2), cultures were fed differentiation media containing 0, 25 or 50 microM DHEA for 20 d. At the end of this treatment period, cultures were either prepared for lipid staining using Oil Red O or for marker enzyme analysis (glycerol-3-phosphate dehydrogenase activity; GPDH). To determine if the stimulatory effects of DHEA on SV cell differentiation were dependent on the presence of thiazolidinediones (Experiment 3), cultures of differentiating SV cells were incubated in the presence and absence of BRL 49653 and either 0, 25 or 50 microM DHEA. In Experiment 1, cultures treated with 25 and 100 microM DHEA had fewer cells than cultures treated with either 0 or 5 microM DHEA. Alamar Blue staining decreased as the level of DHEA in the cultures increased. In Experiment 2, cultures treated with DHEA had more lipid and GPDH activity than control cultures. In Experiment 3, cultures treated with BRL 49653 had more triglyceride than cultures treated without BRL 49653. Likewise, cultures treated with DHEA had more triglyceride than their non-DHEA controls. Regardless of the BRL status, cultures supplemented with DHEA had more triglyceride than control cultures. These data suggest that in cultures of SV cells from human adipose tissue, DHEA supplementation attenuates proliferation and enhances differentiation. These data support the hypothesis that DHEA directly attenuates preadipocyte proliferation in humans as we previously demonstrated in primary cultures of pig and rat SV cells and in cultures of 3T3-L1 preadipocytes. In contrast, DHEA stimulated the differentiation of human preadipocytes, which is contrary to its actions in differentiating cultures of preadipocytes from animals.

Expression of CCAAT/Enhancer Binding Proteins during Porcine Preadipocyte Differentiation

The expression of three CCAAT/enhancer-binding proteins (C/EBPs) was examined with immunocytochemistry and Western blot analysis during preadipocyte differentiation in porcine stromal vascular (S-V) cell cultures. Regardless of treatment and time in culture, immunoreactivity for all three C/EBP isoforms was restricted to cell nuclei. At day 1, 50 ± 6% of S-V cells were C/EBPδ positive, whereas 13 ± 3 and 11.7 ± 3% of S-V cells were AD-3 and C/EBPα positive, respectively. After 3 days of seeding in fetal bovine serum (FBS) and dexamethasone (DEX), C/EBPδ; AD-3, and C/EBPα-positive cells increased to 67 ± 5, 42 ± 4, and 32 ± 3%, respectively. Double staining clearly showed that most of the C/EBPα reactive cells had not accumulated appreciable lipid after 3 days of FBS + DEX. Following 3 days of insulin treatment, the percentage of C/EBPδ cells was 50 ± 6, whereas the percentage of AD-3- and C/EBPα-positive cells was 41 ± 4 and 31 ± 3, respectively. After insulin treatment all fat cells were AD-3, C/EBPα, and C/EBPδ positive. Double staining demonstrated that fat cells were C/EBPδ reactive throughout the culture period. Western blotting showed changes in C/EBP isoform expression that were consistent with the immunocytochemical results. We conclude that C/EBPα is a terminal differentiation marker which is expressed later than AD-3 but further studies are needed to determine the relationship between C/EBPδ and adipogenesis in porcine S-V cultures.

Dehydroepiandrosterone attenuates preadipocyte growth in primary cultures of stromal-vascular cells.

The purpose of this study was to determine whether the antiobesity actions of dehydroepiandrosterone (DHEA) are due to an influence on preadipocyte proliferation and/or differentiation in primary cultures of pig and rat stromal-vascular (SV) cells. Pig SV cells were isolated from dorsal subcutaneous adipose tissue of 7-day-old pigs. For the proliferation assays, pig SV cells were grown for 4 days in plating medium containing DHEA at 0, 15, 50, or 150 microM. For the differentiation assays, pig SV cells were grown in plating medium for 3 days and then switched to a serum-free medium containing DHEA at 0, 15, 50, or 150 microM for the next 6 days. Rat SV cells were isolated from inguinal fat pads of 5-wk-old male rats. Rat SV cells were exposed to DHEA at 0, 5, 25, or 75 microM during proliferation. For the differentiation assays, rat SV cells were grown for 8 days in a serum-free medium containing DHEA at 0, 5, 25, or 75 microM. Preadipocyte differentiation [lipid staining, glycerol-3-phosphate dehydrogenase (GPDH) activity] and proliferation (preadipocyte-specific antigen staining) decreased with increasing levels of DHEA in cultures of pig SV cells. In cultures of rat SV cells, preadipocyte differentiation (lipid staining, GPDH activity) and proliferation ([3H]thymidine incorporation) were decreased in the 25 and 75 microM DHEA groups compared with the control and 5 microM DHEA groups. The level of expression of CCAAT enhancer binding protein-alpha, a master regulator of adipogenesis, in cultures of pig SV cells treated with 150 microM DHEA was 38% of control cultures. These data support the hypothesis that DHEA directly attenuates adipogenesis via attenuation of preadipocyte proliferation and differentiation.

Preadipocyte screening by laminin in porcine stromal vascular cell cultures.

To determine if subpopulations of cells in stromal vascular (S-V) cultures could be segregated and separated based on affinity for laminin substratum. S-V cells were seeded and allowed to attach for various times; 4 hours was found to be optimal for cell attachment. Cultures were rinsed after 4 hours of seeding, and S-V cells were divided into three subpopulations based on affinity for laminin: (1) cells that did not attach to laminin; (2) cells that had a low affinity for laminin; and (3) cells that had a high affinity for laminin. After 24 hours, cultures were either stained for the AD-3 antigen (a marker for preadipocytes), C/EBP-alpha (a terminal differentiation marker), or C/EBP-delta (an early preadipocyte marker). Companion cultures were treated with various media for 9 days and stained with oil red-O. Cells with a high affinity for laminin had the highest proportion of AD-3 and C/EBP-alpha positive cells and the highest proportion of fat cells after treatment with insulin +/- dexamethasone. Cells with a low affinity for laminin had the highest proportion of C/EBP-delta cells and the highest proportion of fat cells after treatment with fetal bovine serum+dexamethasone, followed by insulin. These results indicate that differentiating preadipocytes adhere to laminin to a much greater degree than do non-preadipocytes. Therefore, laminin-coated dishes can be used to screen S-V cells to produce preadipocyte or fibroblast-enriched S-V cultures.

Newly recruited and pre-existing preadipocytes in cultures of porcine stromal-vascular cells: morphology, expression of extracellular matrix components, and lipid accretion.

Expression of extracellular matrix (ECM) components during differentiation of pre-existing preadipocytes and preadipocytes recruited by dexamethasone (DEX) was examined with immunocytochemistry in primary cultures of adipose tissue stromal vascular (S-V) cells. Immunocytochemistry showed that a small proportion of preadipocytes (AD-3+) in 24-h cultures (d 0 to 1) contained lipid or expressed ECM. Two days of insulin treatment markedly increased preadipocyte ECM expression, and preadipocytes were "rounder" than those not treated with insulin. Dexamethasone with insulin increased preadipocyte recruitment two- to fivefold in completely serum-free cultures and in cultures serum-free after seeding and plating in serum for 1 to 3 d. Double staining demonstrated that ECM expression and lipid accretion were tightly coupled and lagged significantly behind preadipocyte recruitment (AD-3 expression). Double staining (lipid and AD-3) also demonstrated remarkable and unexpected cytological traits indicating a "reticuloendothelial" nature of newly recruited preadipocytes. Time-lapse phase contrast microscopy verified these observations and demonstrated that small adipocytes and preadipocytes migrated and formed cell-to-cell contacts while aggregating and clustering. Large clusters of lipid-free preadipocytes developed in DEX-treated cultures, but not in cultures treated with DEX + insulin. However, the influence of DEX on preadipocyte recruitment and ECM expression was independent of insulin. Preadipocytes on ECM substrata accumulated lipid but were "flat" and did not express ECM components, regardless of insulin or DEX treatment. These studies clearly indicate that preadipocytes express ECM components after recruitment, and the ECM may be critical for morphological development of adipocytes.

The interaction between vitamin A and thiazolidinedione on bovine adipocyte differentiation in primary culture.

We studied the effects of a thiazolidinedione (T-174) and retinoids on adipocyte differentiation in beef cattle. Stromal-vascular (SV) cells containing preadipocytes were prepared from perirenal adipose tissue of 21-mo-old Japanese Black steers. After confluence, these cells were cultured in 25 microM T-174, which is a specific ligand for an adipogenesis stimulating nuclear receptor (i.e., gamma subtype of peroxisome proliferator-activated receptor [PPARgamma]), with 1 microM all-trans retinoic acid (RA) or .4 to 40 microg/100 mL of retinol for 10 to 14 d. The number of cells accumulating lipid droplets was counted as morphologically differentiated adipocytes, and the activity of glycerol-3-phosphate dehydrogenase (GPDH), a biochemical index for the differentiation, was determined. The number of lipid-laden cells and GPDH activity were increased by the addition of T-174. All-trans retinoic acid completely blocked the stimulative action of T-174. The addition of retinol also decreased the number of lipid-laden cells and GPDH activity in a dose-dependent manner. These results showed that the thiazolidinedione stimulated adipocyte differentiation and the retinoids blocked the adipogenesis induced by the thiazolidinedione in primary culture of bovine SV cells. Peroxisome proliferator-activated receptor gamma may play an important role in adipocyte differentiation, and retinoids may interfere with the action of PPARgamma in cattle.

Differentiation-dependent expression of obese (ob) gene by preadipocytes and adipocytes in primary cultures of porcine stromal-vascular cells

The relationship between obese (ob) gene expression and preadipocyte differentiation was examined in primary cultures of porcine stromal-vascular (S-V) cells by Northern-blot analysis using a pig ob cDNA probe. Isolated adipocytes expressed high levels of ob gene, but S-V cells did not express the ob gene. Cultures were seeded with fetal bovine serum (FBS) plus dexamethasone (Dex) for 3 days followed by ITS (insulin 5 μg/ml, transferrin 5 μg/ml, and selenium 5 ng/ml) treatment for 6 days. Detectable levels of ob mRNA first appeared at day 1 with very low activity of glycerol phosphate dehydrogenase (GPDH). Levels of ob mRNA increased in parallel with preadipocyte number or GPDH activity at the later times in cultures. The depletion of preadipocytes by complement-mediated cytotoxicity at day 3 of culture resulted in markedly decreased ob mRNA expression. Immunocytochemical analysis showed that ob protein was localized in the cytosol of preadipocytes and adipocytes. These data indicated that the ob gene is expressed by preadipocytes and ob gene expression may be correlated with preadipocyte recruitment as well as fat cell size.

Preadipocyte recruitment in stromal vascular cultures after depletion of committed preadipocytes by immunocytotoxicity.

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 +/- 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 +/- 5.1/ unit area) after removal of preadipocytes. Immunocytochemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 +/- 45/unit area) than FBS alone (10.5 +/- 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation; and that DEX induced early preadipocyte differentiation depends on mitosis.