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The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte differentiation and insulin-like growth factor binding protein (IGFBP) secretion in stromal-vascular (S-V) cell cultures established from subcutaneous adipose tissue obtained from nine 75 day and four 50 day pig fetuses. Cultures of S-V cells from four young pigs (5-7 days old) were also studied. Each fetal S-V cell culture represented 1 pool of S-V cells/dam. Cultures were seeded and plated in 10% FBS from day 0-3 and treated with insulin (ITS) + 10 nM DEX from day 3-6 (late DEX treatment). Alternatively, cultures were seeded and plated in 10% FBS + 80 nM DEX from day 0-3 and treated with insulin alone from day 3-6 (early DEX treatment). Conditioned media was collected on day 6 of culture after 3 days of conditioning, and prepared for subsequent 125I-IGF-I ligand blot analysis for IGFBPs and RIA for IGF-I and IGF-II. Early and late DEX increased (P<0.05) preadipocyte (AD-3+) recruitment but only early DEX increased preadipocyte differentiation (lipid + and C/EBP alpha+) by day 6 in S-V cultures from 75 day fetuses. Levels of IGFBP-2, IGFBP-4, IGF-I and IGF-II in media conditioned by 75 day fetal S-V cultures were not influenced by late DEX. However, late DEX reduced levels of 29 kDa IGFBPs and markedly increased (P<0.05) IGFBP-3 levels in 75 day S-V media. Late DEX also markedly increased (P<0.05) IGFBP-3 levels in 50 day S-V media but had little influence on other IGFBPs. Early DEX treatment increased (P<0.05) IGFBP-4 levels in 75 day S-V media but had little to no influence on levels of IGF-I, IGF-II and other IGFBPs. These studies indicate that IGFBP-4 may regulate local metabolism during preadipocyte differentiation, whereas IGFBP-3 may antagonize preadipocyte differentiation by targeting IGF-I away from differentiating cells and towards growing cells.

The present study examined the influence of fetal age and thyroxine (T4) and growth hormone (GH) treatment, on the expression of insulin-like growth factor binding proteins (IGFBPs) in fetal pigs. On day 70 of gestation fetuses were either hypophysectomized (hypox), hypox and implanted with T4 pellets, or left intact, and were recovered 5, 10, 15 and 20 days following hypox and T4 pellet placement. Intact fetuses were also recovered from several dams at 50 days of gestation. In additional dams, hypox fetuses (day 70) were implanted with GH loaded Alzet mini-pumps on day 90, and control, untreated, and GH-treated hypox fetuses were recovered on day 105 of development. Subcutaneous adipose tissue, serum and other fetal tissues were collected at the time of recovery and prepared for subsequent ligand blot analysis with 125I -IGF-1 and immunoblot analysis with IGFBP antibodies. The main effect of IGFBP was significant (P <0.01) for age associated changes in serum IGFBP percentages. Between 50 and 75 days of fetal development the levels of 29 kDa IGFBPs in adipose tissue and serum markedly increased. In contrast, IGFBP-2 levels decreased and IGFBP-4 levels increased in adipose tissue while IGFBP-2 levels increased and levels of IGFBP-4 and -3 decreased in serum. Fetal hypox decreased adipose tissue IGFBP levels in a time and IGFBP-dependent manner. For instance, IGFBP-2 and 29 kDa IGFBP levels decreased much faster after fetal hypox than did IGFBP-3 levels whereas IGFBP-4 levels did not decrease. The main effect of IGFBP was significant (P<0.01) for T4-induced changes in adipose tissue IGFBP levels. T4 treatment increased adipose tissue levels of 29 kDa IGFBPs but did not influence IGFBP-2,-3 and -4 levels. GH treatment had no influence on adipose tissue or serum IGFBP levels. These studies indicate that IGFBP-1 (one of the 29 kDa IGFBPs) may be the major IGFBP mediator of the influence of T4 on fetal development.

At 90 days of gestation hypophysectomized (hypox) porcine fetuses were treated with hydrocortisone (HC) or thyroxine (T4) and S-V cultures prepared from intact, treated and untreated hypox fetuses at 105 days of gestation. After 24 h, cultures were stained for the AD-3 antigen and the transcription factors, C/EBP alpha and delta. The proportion of preadipocytes (AD-3+) was doubled in cultures derived from HC treated hypox fetuses compared to cultures from intact, untreated and T4 treated hypox fetuses. Compared to cultures from intact fetuses, proportions of C/EBP delta reactive cells (approximately 40%) were increased similarly in cultures from untreated and HC treated hypox fetuses. Compared to day 0-1, preadipocyte proportions were markedly reduced in cultures from untreated and HC treated fetuses and increased in cultures from intact and T4 treated fetuses after three days of fetal bovine serum (FBS; day 0-3). After six days of insulin (FBS day 0-3-->insulin, transferrin and selenium; ITS), fat cell numbers were 5 fold higher in cultures from T4 treated fetuses compared to cultures from untreated and HC treated hypox fetuses. Other cultures were seeded and plated in FBS + 80 nM dexamethasone (DEX; day 0-3) followed by insulin (day 3-9). Preadipocyte development was independent of the source of S-V cells in cultures treated with FBS + DEX-->insulin. These data indicate that, compared to preadipocyte recruitment, hormonal regulation of adipocyte differentiation may be more complex and involve more interactions.

The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures. Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting. After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls. Anti-IGF-1 decreased fat cell proportions by 14-fold. Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold. Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively. TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment). There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls. Control antibodies and negative controls had no effect. These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.

The effects of fetal hypophysectomy (hypox) and age on insulin-like growth factor-I (IGF-I) response, receptor binding and IGF-I & IGF binding protein secretion by porcine preadipocytes was examined. Primary cultures of stromal vascular (S-V) cells derived from fetal and postnatal adipose tissue were established and exposed to test media (different concentrations of pig serum with or without IGF-I) for morphological study or to serum-free media for measuring IGF-I receptor binding and IGF-I & IGFBPs secretion. Conditioned media were collected and subjected to IGF-I RIA and ligand blot and immunoblot analysis. IGF-I significantly stimulated total cell growth (determined by DNA levels) and increased fat cell cluster number and lipid deposition in porcine S-V cultures (determined by image analysis). Although the pattern of response to IGF-I was similar in fetal and postnatal cultures, the response was greater in fetal cultures. The mitogenic and adipogenic response of fetal preadipocytes to IGF-I was impaired by fetal hypox. Specific IGF-I binding sites on fetal preadipocytes had a higher binding affinity for IGF-I (Kd = 2.82 +/- 0.28 nM) than did postnatal preadipocytes (Kd = 7.28 +/- 1.52 nM, P < 0.05). Preadipocytes secreted IGF-I and four IGF-BPs (BP-1, -2, -3, and -4) in a developmentally regulated manner. For example, IGFBP-2, and -3 were predominant binding proteins secreted by fetal preadipocytes which represented 47% and 30% of the total, respectively. IGFBP-1 was the major binding protein secreted by postnatal preadipocytes. Furthermore, fetal cells secreted greater amounts of IGF-I in cultures than did postnatal cells (2428 +/- 205 pg/ml vs. 870 +/- 31 pg/ml, P < 0.05). Fetal hypox caused a decrease from 47% to 68% of control values in all four IGFBPs secreted by preadipocytes. The changes in IGF-I receptor binding and IGF-I and IGFBP secretion by preadipocytes may account for altered response of preadipocytes to IGF-I. Thus, secretion of IGF-I and IGFBPs, IGF-I binding and response of porcine preadipocytes were affected by age and fetal hypox. The results indicated that locally produced IGF-I and IGFBPs may play a critical paracrine/autocrine role during fetal adipose development.

Studies (in situ hybridization) were performed on the developing subcutaneous tissue (SQ) of the pig fetus to determine sites of synthesis of TGF-beta 1 and IGF-II. Tissues from 50, 70, 90, 110-day-old pig fetuses and 7-day-old postnatal pigs were Bouin's fixed, paraffin embedded and hybridized with biotin labelled cDNA probes. The expression of TGF-beta 1 messenger RNA was detectable primarily in the dermis, hair follicle fat lobule, outer and inner SQ areas at all ages. Cells adjacent to adipocyte clusters and some small adipocytes were positive for TGF-beta 1. There was no positive hybridization of TGF-beta 1 probes in the developing muscle below the SQ. The expression of IGF-II was evident in the developing muscle below the SQ at 50 d and 70 d, and could be detected in the outer and inner SQ at 70 d. Much lower levels of IGF-II expression were observed in 90 and 110 d fetuses, but an increase in IGF-II expression was evident in the muscle of 7d postnatal pigs. Our results suggest that paracrine and autocrine regulatory systems may be operative for developing adipocytes (TGF-beta 1) and muscle (IGF-II), with specific areas and times of TGF-beta 1 and IGF-II expression being evident in developing pig SQ tissue.

An ontogeny study of lean and pre-obese pig fetuses was conducted to evaluate the temporal and genetic influence on the relationship between growth and IGF production. At 70, 90 and 110 days of gestation, three fetal pigs were obtained from each of three obese and lean dams. The heart, kidneys, lungs, liver and one biceps femoris muscle were removed, weighed, frozen and extracts prepared. For all the organ and tissue weights, there were significant (P less than .001) main effects of genotype (G) and fetal age (A). At each fetal age, pre-obese fetuses had lighter organ and tissue weights. The content of IGF's in serum and various tissues and/or organs was also determined. Main effects of A and G were significant (P less than .05) for liver and lung IGF-I and muscle IGF-II concentrations. Main effects of G were significant (P less than .01) for liver IGF-II whereas muscle IGF-I and lung IGF-II concentrations were only affected by fetal age (P less than .01). Overall, liver IGF-I and II and muscle IGF-II concentrations were higher in lean fetuses (P less than .01). Serum IGF-I concentration increased (P less than .01) with fetal age and was independent of fetal strain. Serum IGF-II concentration increased with age (P less than .01) in lean but not pre-obese fetuses. Therefore, these studies demonstrate genotype dependent growth factor concentrations in tissues and organs of developing fetuses. Finally, serum concentration of IGF's were strongly correlated (positive) with overall growth but were not strongly correlated with tissue and organ concentration of IGFs.