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Objective: 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., Research Methods and Procedures: 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., Results: 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., Discussion: 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.

Objective: This study compares the effects of the thiazolidinedione, troglitazone (TGZ), dexamethasone (DEX), and DEX plus TGZ on preadipocyte differentiation and the expression of CCAAT/enhancer-binding protein-alpha (C/ EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma)., Research Methods and Procedures: Adipose tissue was obtained from postnatal pigs to isolate stromal-vascular cells. First, we applied 1, 5, or 10 microM TGZ and 10% fetal bovine serum for 3 days and counted the number of recruited preadipocytes. Next, we used either 10 microM TGZ, 80 nM DEX, or DEX plus TGZ with 10% fetal bovine serum for 3 days and then switched to serum-free medium with insulin for 6 days. On day 3 of culture, we counted preadipocytes, and on days 3 and 6 of culture, we performed immunostaining and Western blot analysis to determine the expression of C/EBPalpha and PPARgamma proteins. On day 9 of culture, we stained for lipids with oil red-O and measured the activity of glycerol phosphate dehydrogenase., Results: DEX and TGZ equally enhanced recruitment of preadipocytes and late differentiation, but these effects were not additive with DEX plus TGZ treatment. However, TGZ and DEX had a differential effect on morphogenesis; DEX-treated adipocytes were larger and organized in loose clusters, whereas TGZ-treated cells were smaller and formed compact clusters. Both agents increased C/EBPalpha expression but in a temporally distinct manner. DEX was a better inducer than TGZ, and its effect was early and temporary. However, treatment with either TGZ or DEX did not change PPARgamma protein expression as evaluated by a Western blotting, but immunocytochemistry showed a tendency for increased numbers of PPARgamma positive cells., Discussion: TGZ and DEX equally enhance early and late adipocyte differentiation, possibly by using some common pathways for preadipocyte recruitment. The differential effect on morphogenesis implies a potential differential effect on the expression of extracellular matrix components. C/EBPalpha may be the critical transcription factor involved in TGZ- and DEX-induced adipogenesis.

Objective: 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., Research Methods and Procedures: 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., Results: 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)., Discussion: 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.

Objectives: 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., Research Methods and Procedures: Freshly isolated S-V cells from rat inguinal adipose tissues were plated for 24 hours then exposed to serum-free medium., Results: 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., Discussion: 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.

Objective: This study tested the hypothesis that combined treatment of thyroxine (T4) and growth hormone (GH) could normalize cellular and metabolic aspects of adipose tissue development of hypophysectomized fetal pigs., Research Methods and Procedures: On day 70 of gestation, pig fetuses were hypophysectomized by microcauterization or remained intact. Hypophysectomized fetuses remained untreated or were treated from day 90 to day 105 of gestation with T4, GH, or a combination of both hormones., Results: Body weights were unaffected by hypophysectomy or hormone treatment. De novo lipogenesis in subcutaneous adipose tissue was increased 10-fold by hypophysectomy, consistent with our previous results. This increase was abolished by GH treatment in the hypophysectomized fetuses. In contrast, T4 treatment of the hypophysectomized fetuses resulted in a 12-fold further increase in adipose tissue lipogenesis, an effect that was negated by concomitant administration of GH. Lipolytic response to isoproterenol was decreased by hypophysectomy, unaffected by GH treatment, and restored to intact values by T4 or by T4+GH treatment in the hypophysectomized fetuses., Discussion: In contrast to T4, GH does not influence serum insulin-like growth factor-I or adipose tissue lipolysis, but decreases lipogenesis in the fetal pig. However, replacing both T4 and GH normalized hypophysectomized fetuses to a greater extent than either GH or T4 alone. Thus, any influence of thyroid hormones on stimulating adipose tissue lipogenesis in the developing fetal pig may be normally counterregulated by pituitary-derived growth hormone.

Objective: To determine if subpopulations of cells in stromal vascular (S-V) cultures could be segregated and separated based on affinity for laminin substratum., Research Methods and Procedures: 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., Results: 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., Discussion: 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.

The hormonal regulation of leptin mRNA expression and the association between leptin expression and adipocyte differentiation were examined in primary cultures of porcine S-V cells with Northern blot and immunocytochemical analysis. Seeding for 3 days with fetal bovine serum (FBS) with varying levels of dexamethasone (Dex) increased levels of leptin mRNA in a dose-dependent manner in parallel with increases in the proportion of preadipocytes (AD-3 positive cells; AD-3, a preadipocyte marker). Six-day treatment with 10 or 850 nM insulin after FBS+Dex treatment resulted in a similar increase in leptin mRNA expression and morphological differentiation. However, significantly lower levels of leptin mRNA and smaller fat cells were observed in cultures treated with 1 nM insulin or 10 nM insulin-like growth factor-I (IGF-I). Dex-induced increases in leptin mRNA levels and AD-3 cell numbers were blocked completely by the addition of transforming growth factor-beta (TGF-beta) to FBS+Dex-treated cultures. However TGF-beta significantly increased fat cell size and leptin mRNA expression when added to ITS (insulin, 850 nM; transferrin, 5 microg/ml; and selenium, 5 microg/mL) treated cultures during the lipid-filling stage. When added with FBS+DEX for the first 3 days, growth hormone (GH) did not influence the Dex-induced increase in AD-3 cells and leptin mRNA expression, but GH reduced leptin mRNA levels when added with insulin for 6 days after FBS+Dex. These results demonstrated that regulation of leptin mRNA expression by Dex, insulin, IGF-I, TGF-beta, and GH may be associated with changes in preadipocyte number and fat cell size.

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.

Studies were conducted to determine the influence of thyroxine (T4) in vivo on preadipocyte development and insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) secretion in stromal-vascular (S-V) cultures. Fetal pigs were hypophysectomized (hypox) at 70 days of gestation, implanted with T4 pellets, and fetuses from the dam at 75 days of gestation. In a second experiment, hypox and T4 implantation were performed at 75 days and fetal pigs removed at 95 days of gestation. Primary cultures of stromal vascular (S-V) cells derived from fetal adipose tissue were established. Cultures were stained for morphological analysis and conditioned media were collected for IGF-1 determination by radioimmunoassay (RIA) and IGFBP analysis by Western blotting. After only 5 days of T4 treatment, fat cell cluster number and size and lipid deposition in cultures were significantly increased compared to cultures from untreated hypox fetuses. Fetal hypox reduced IGF-I secretion by preadipocytes at both ages and T4 treatment completely normalized IGF-I secretion (p < 0.05). Four IGFBPs (BP-1, BP-2, BP-3 & BP-4) detected in S-V cultures derived from 95-day fetuses were decreased in concentration by hypox by 44 +/- 9.4%, 32 +/- 9.7%, 42 +/- 12% and 53 +/- 6.9%. In cultures derived from T4 treated hypox fetuses, the levels of these four IGFBPs were increased by 187 +/- 25%, 239 +/- 38%, 190 +/- 5% and 347 +/- 43% over control values, respectively. In cultures from 75-day fetuses, only IGFBP-2 (major one) and BP-1 (minor one) were detected and their secretion was also decreased by hypox and elevated by T4 treatment (190 +/- 49.5%, 156 +/- 30%, respectively, of controls). The results provide direct evidence that T4 has a major influence on fetal preadipocyte development. T4 stimulated production of IGF-I and IGFBP in fetal S-V cultures, which in turn, may have mediated the capability of T4 to enhance fetal adipose tissue development.

The temporal aspect of thyroxine (T4) enhanced development of several fetal adipose tissue depots (locations) and skin was examined in the present study. On day 70 of gestation pig fetuses were either hypophysectomized (hypox), hypoxed and implanted with T4 pellets or left intact (control). Subsequently, fetuses were removed for study at either 73, 75, 80, 85 and 90 days of gestation. Serum levels of T4 and insulin-like growth factor I (IGF-I) were significantly elevated by T4 after 3 days of treatment and throughout the study. T4 treatment enhanced development of skin, inner and outer subcutaneous (SQ) adipose tissue and adipocyte lobules around hair follicles (HFL's) but degree of response and response time were tissue or depot dependent. The T4 response time was shortest for skin and HFL's, longest for inner SQ and intermediate for the outer SQ. Considering the entire treatment period, magnitude of the T4 response was much greater for skin and HFL's than for outer and inner SQ tissues. Possibly, these developmental characteristics indicate that T4 directly influences skin and HFL's and indirectly influences inner SQ adipose tissue by increasing production of a locally produced adipogenic growth factor. Furthermore, developmental characteristics (morphological) indicated that putative local growth factor mediation of T4 action may depend on stage of development or maturity of the tissue. IGF-I is clearly the most logical candidate as a putative local mediator of T4, but evidence at the molecular level is needed to prove this suggestion. Regardless, the particular adipose tissue location or depot is an important aspect of hormone responsiveness during the critical or sensitive period of fetal adipose tissue development.

Monoclonal antibodies (MAbs) raised against porcine adipocyte plasma membranes were used to demonstrate complement-mediated cytotoxicity of adipocytes and preadipocytes in primary stromal-vascular (SV) cultures. Five of the six MAbs tested significantly reduced the number of fat cell clusters in cultures maintained in medium supplemented with pig serum and dexamethasone (PS/DEX) but not in cultures supplemented with insulin, transferrin, and selenium (ITS). Neither MAb nor complement alone affected fat cell cluster number. Treatment of both ITS and PS/DEX cultures with pools of 2 or more MAbs, in combination with complement, eliminated fat cell clusters in all instances. Treatment of cultures prior to appearance of cells containing lipid demonstrated that preadipocytes, or adipose lineage cells, could also be eliminated by MAb/complement treatment. Finally, injection of young rats with a pool of three of the MAbs produced a 30% reduction in inguinal fat pad weight without affecting other tissues. Adipocyte/preadipocyte-depleted cultures can now be used as a model system to examine progression of cells through the adipose cell lineage at a time not previously possible with primary cells.

Elevated serum hydrocortisone (HC) levels are associated with larger fat cells and elevated levels of lipogenic and associated enzymes in late term pig fetuses from genetically obese dams. We have investigated the influence of HC status per se on these and other adipose tissue traits by chronically treating pig fetuses hypophysectomized (hypox) on day 70 with HC between either day 70 and 90 or 90 and 105 of gestation. Treatment with HC during both periods increased serum HC levels (P<.05) and increased fat cell size (P<.05) in the perirenal (PERI) and subcutaneous (SQ) depots, but failed to influence body weights, insulin-like growth factor-1 and insulin levels. Quantitative analysis of sections of PERI and SQ adipose tissue indicated that HC increased lipoprotein lipase (LPL), esterase and glucose-6-phosphate dehydrogenase (G6PDH) activities. The degree of esterase and G6PDH, but not LPL response to HC, was greater during the 90- to 105-day period than during the earlier period. HC significantly increased lipid accretion only in the SQ depot between 90 and 105 days. Overall, HC significantly augmented hypox-induced alterations in cellular and metabolic traits of developing adipose tissue. The general increase in fat cell size (21%) with moderate (SQ-105d) or no (PERI-90, 105d; SQ-90d) increase in lipid accretion indicates that HC either did not influence or decreased apparent fat cell number. Regardless, these data indicate that changes in serum HC per se may account for adipose tissue traits that characterize fetuses from genetically obese dams.

Glucocorticoids have been shown to be essential for the excessive fat deposition and development of obesity in several animal models. This study was performed to characterize the role of glucocorticoids in the developmental regulation of adipose tissue metabolism. On day 70 of gestation, pig fetuses were hypophysectomized by micro-cauterization. Hypophysectomized fetuses were implanted subcutaneously with hydrocortisone pellets or received no hormone replacement. Fetuses were removed by laparotomy on day 90 of gestation. Additional fetuses were hypophysectomized on day 70, implanted with hydrocortisone pellets on day 90 and removed on day 105 of gestation. Several intact fetuses were also implanted subcutaneously with hydrocortisone pellets during this later gestational period. Serum cortisol concentrations were reduced in hypophysectomized pigs at both fetal ages and were restored to intact levels by hydrocortisone treatment. Hydrocortisone supplementation enhanced lipolytic response to isoproterenol in intact fetuses but failed to restore lipolytic response to isoproterenol in hypophysectomized animals at either fetal age. Hydrocortisone induced a slight increase in lipogenesis in hypophysectomized fetuses when administered from 70 to 90 days of gestation and a more dramatic increase when administered from days 90 to 105 of gestation. However, hydrocortisone had no effect on basal or insulin stimulated lipogenesis in intact fetuses when administered from days 90 to 105 of gestation. These results indicate that hydrocortisone may have a primary influence on adipose tissue metabolism during late fetal development only in the absence of inhibition from counterregulatory hormones of pituitary origin.

Late term fetuses from genetically obese dams have slightly larger fat cells, greater adipose tissue lipoprotein lipase (LPL) activities, elevated levels of thyroid hormones, and depressed growth hormone (GH) levels when compared to fetuses from lean dams. We have investigated the influence of thyroid hormone and GH status per se on these and other adipose tissue traits by chronically treating hypophysectomized (hypox) fetuses (day 70) between day 90 and 105 of gestation with either thyroxine (T4) or human GH. Treatment with T4 decreased body weights (P<.05), increased serum T4 levels (P<.05), and enhanced skin and hair development (P<.05). Quantitative analysis of sections of perirenal and subcutaneous adipose tissue indicated that T4 increased LPL activity (P<.05), slightly increased fat cell size, and more than doubled (P<.05) lipid accretion. A hypox induced deficit in fat cell cluster number in the outer layer of subcutaneous tissue was normalized by T4 (P<.05). Conversely, human GH (hGH) treatment had no influence on body weight, increased serum hGH levels, decreased fat cell size (P<.05) and LPL activity (P<.05) but had no influence on lipid accretion. Quantitative analysis of adipose tissue sections provided direct and indirect evidence of a "critical" or "sensitive" period between 90 and 105 days, since fetal hypox at day 70 severely impeded preadipocyte recruitment/replication during this period. Furthermore, T4 but not GH effectively normalized this hypox-induced deficiency in preadipocyte development. Therefore, T4 may have a major role in preadipocyte recruitment/replication during late fetal life.