Recruitment and differentiation of intramuscular preadipocytes in stromal-vascular cell cultures derived from neonatal pig semitendinosus muscles.
ABSTRACT 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.
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ABSTRACT: Previous studies have shown that the responsiveness of porcine preadipocytes to glucocorticoids increases progressively with fetal age. In the current study, the development of fetal glucocorticoid receptors in porcine preadipocytes was examined in primary cultures derived from 50-, 75-, and 105-d fetal and 7-d postnatal porcine adipose tissue. Three dams were used for each fetal age group and three young pigs were used for the postnatal studies. Using [3H]dexamethasone as the radioligand, cytosolic glucocorticoid receptors were measured in preadipocytes. There was an age-related increase in the number of glucocorticoid receptors during fetal development. The number of glucocorticoid receptors was very low in 50-d fetal porcine preadipocytes (1.350 +/- .178 fmol/mg of protein) and increased progressively to a peak at 105 d (8.990 +/- .741 fmol/mg of protein). This represents an approximate sixfold increase in receptor number between these two ages (P < .05). After birth, the glucocorticoid receptor number significantly decreased compared with that of 105-d fetuses (2.766 +/- .218 fmol/mg of protein, P < .05). Furthermore, the dissociation constant (Kd) of the glucocorticoid receptor in 105-d fetal preadipocytes was numerically smaller than the Kd of receptors derived from other age groups, although only significantly lower than that of the 75-d group (P < .05). Insulin markedly increased the number and binding affinity of glucocorticoid receptors in preadipocytes, indicating its potential involvement in regulation of glucocorticoid receptors. The effect of glucocorticoids on differentiation in preadipocytes is, at least in part, mediated by the number of glucocorticoid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)Journal of Animal Science 04/1995; 73(3):722-7. · 2.09 Impact Factor
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ABSTRACT: In order to study how adipose conversion affects the extracellular environment, levels of extracellular matrix (ECM) proteins during differentiation were analyzed by 125I-labeled antibody binding to each specific primary antibody. When confluent bovine intramuscular preadipocytes (BIP) were stimulated with adipogenic medium, there was a significant accretion on the cell surface of type I-VI collagens, laminin and fibronectin, compared with undifferentiated cells. The deposition amount of ECM proteins had reached near maximal levels at an early stage of differentiation and lasted throughout the culture. However, the increasing manners were not all the same in these eight proteins. Type V and type VI collagen tended to show a transient decline after the rapid rise at the beginning of stimulation, and fibronectin instead, subsequently decreased. Further analysis by immunocytochemical staining showed that remodeling occurred in type V and VI collagen matrices during this period; extensive fibrillar networks seen at 10 d after stimulation were quite unlike that formed earlier. These specific increases and development of matrix during adipocyte differentiation imply some significance for organizing fat lobules in each ECM proteins, especially type V and VI collagens.Biology of the Cell 07/2002; 94(3):197-203. · 3.49 Impact Factor
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ABSTRACT: 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.Journal of Animal Science 06/2000; 78(5):1227-35. · 2.09 Impact Factor