Glucocorticoid hormones contribute to the adipogenic activity of human serum

The adipose conversion of 3T3-L1 fibroblasts depends on serum factors, one of which has been identified as GH. Human serum extracts obtained by heat treatment do not contain intact GH, as shown by RIA, but still support adipose conversion. After organic solvent extraction followed by reversed phase HPLC, two adipogenic fractions can be purified. Their identification as cortisol and cortisone follows from the observations that both glucocorticoid hormones are strongly adipogenic in physiological concentrations and elute upon reversed phase HPLC with retention times identical to those of the adipogenic fractions. After derivatization by acetylation, the adipogenic fractions purified from human serum again do not differ from C-21 monoacetyl cortisol and -cortisone with regard to adipogenic activity and behavior on reversed phase HPLC and TLC. Treatment of heat extracts from human serum with cortisol antibodies strongly reduced their adipogenic activity which could be restored only after addition of cortisol to the original concentration. We conclude from these results, that the adipogenic activity of human serum depends mainly on its glucocorticoid (as well as GH) concentration.     

Contribution of growth hormone to the adipogenic activity of serum

GH promotes the conversion of cultured preadipose 3T3 cells into adipose cells. The serum of most animals also promotes this differentiation. In order to determine the extent to which GH is responsible for the adipogenic activity of serum, we used a specific antiserum able to suppress completely the adipogenic activity of rat and bovine GH. This antiserum also suppressed all the adipogenic activity secreted into culture medium by a rat pituitary cell line as well as all the adipogenic activity of crude bovine pituitary extract. When tested against the adipogenic activity of serum, the antiserum to GH reduced the activity by one third to half. It is concluded that 1) GH in the concentration and form present in serum is an effective promoter of adipose differentiation, and 2) there exists in serum another adipogenic activity not immunochemically recognizable as GH. This activity is presumed to be of nonpituitary origin.     

Human adipogenic serum activity increases during pregnancy

Adipogenic factor required for adipose differentiation of 3T3-F442A cells is present in human serum. Its activity increases significantly 3-4 fold in maternal serum after the 25th-28th weeks of pregnancy. It remains high until parturition and it is also found at high levels in serum from umbilical cord blood, whereas amniotic fluid from late gestation ages has a very low adipogenic activity. Adipogenic activity from maternal serum decreases abruptly one day after parturition and it reaches normal levels similar to those found in non-pregnant women, remaining constant thereafter. It is suggested that adipogenic serum activity could have some significance for human adipose tissue differentiation.     

Contribution of immune response to the hepatic fibrosis induced by porcine serum

To investigate whether hepatic fibrosis induced by porcine serum in rats is caused by an immune reaction to porcine serum, rats that were immunologically tolerant exclusively to porcine serum were subjected to the repeated injection of porcine serum over a long period. This porcine serum-tolerant group consisted of 15 Wistar rats that had been injected intraperitoneally with porcine serum twice a week from the first postnatal day for 18 weeks. The control group consisted of 16 Wistar rats, aged 8 weeks, that were injected intraperitoneally with porcine serum twice a week for 10 weeks. Livers were fixed and examined by light microscopy. The serum of each rat was subjected to indirect enzyme-linked immunosorbent assay (ELISA) to measure the level of antibody to porcine albumin. In addition, immunohistochemical staining for ED1 was performed on untreated normal and porcine serum-induced fibrotic rat livers to examine the distribution of macrophages and their precursors, the monocytes. All rats in the tolerant group showed an extremely low antibody level (x = 68.27 +/- 4.53), and none (0/15) developed hepatic fibrosis. The majority of rats in the control group showed a very high antibody level (x = 1242.19 +/- 201.15); 75 percent (12/16) developed hepatic fibrosis. Data indicate that, despite the prolonged, repeated injections of porcine serum, if an immune response to porcine serum does not occur, the rats do not develop hepatic fibrosis. The porcine serum-tolerant rats developed hepatic fibrosis after 4 weeks of CCl4 treatment, indicating that injection of porcine serum into neonatal rats did not cause anergy of fibrogenesis, thereby preventing the animal from developing hepatic fibrosis. In normal rat liver, ED1-positive cells, which include nearly all Kupffer cells, were located pre-dominantly in the periportal area. In fibrotic rat liver, ED1-positive cells aggregated prominently in the newly formed and advanced connective tissue septa developed mainly between the neighboring central veins, and in fibrotic parts of the liver capsule. Aggregation of ED1-positive cells was rarely observed in nonfibrotic parts of the liver capsule. The difference between normal and fibrotic rat liver in distribution of EDl-positive cells suggests an involvement of macrophages in fibrogenesis and septum formation. In conclusion, our study showed a significant contribution by the immune response to porcine serum antigens leading to porcine serum-induced rat hepatic fibrosis--processes in which macrophages may be important. This study may lead to an understanding of the mechanism responsible for this form of experimental hepatic fibrosis. (Hepatology 1996 Apr;23(4):811-7)     

Changes in NSILA-S in response to somatotropin administration and hypophysectomy.

The effects on the serum levels of NSILA-S of the administration of human GH (hGH) or of hypophysectomy have been studied. hGH given im (5 mg daily for 3 days) raised the NSILA-S levels of three GH-deficient subjects into the normal range. Significant elevations of NSILA-S were also seen in three normal subjects given im GH. In the same groups of three normal and three GH-deficient subjects, 5 mg hGH administered iv induced an elevation of NSILA-S within 15-60 min. Hypophysectomy in three acromegalics and one subject with a chromophobe adenoma was followed by significant falls of serum NSILA-S. These studies provide further evidence of the dependence of NSILA-S levels on GH.     

Comparison of the mitogenic activity of angiotensin II and serotonin on porcine arterial smooth muscle cells

We have investigated the growth promoting activities of two potent vasoactive substances, serotonin and angiotensin II (AII), on cultured porcine aortic smooth muscle cells (ASMC), using a defined serum-free medium. Serotonin (30 nM to 30 microM) stimulated ASMC DNA synthesis both alone and in combination with platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). Serotonin-induced DNA synthesis was significantly inhibited by ketanserin (5-hydroxytryptamine-2 (5HT-2) receptor antagonist). AII (3-10 nM) failed to stimulate ASMC DNA synthesis directly, either alone or in combination with PDGF or EGF. Since both serotonin and AII were found to activate phosphatidylinositol turnover and are reported to mobilise intracellular calcium, it is apparent that these events alone are insufficient to stimulate ASMC mitogenesis.     

Calf serum modifies the mitogenic activity of epidermal growth factor in WRT thyroid cells

It has already been shown that Wistar rat thyroid (WRT) cells in low concentrations of calf serum (0.5%) are under the influence of both thyrotropin (TSH) and insulin as regards growth. The present data show that epidermal growth factor (EGF), in concentrations up to 10 micrograms/ml, is not able to modify DNA synthesis in WRT cells. On the other hand, insulin-like growth factor I (IGF-I) stimulates DNA synthesis from a dose which is 10-fold lower than that of insulin alone. Combined stimulation of EGF and TSH in WRT cells is equal to that of TSH alone in relation to DNA synthesis, while the combined presence of TSH and IGF-I, or TSH and insulin, in the same medium results in an effect which is greatly superior to the theoretical sum of activities. Repetition of the same experiments using the original clone of WRT cells, but in high concentrations of calf serum (5%), shows that EGF stimulates DNA synthesis in a dose-dependent way from 0.1 to 100 ng/ml. Under these conditions, combined stimulation of EGF with TSH shows that DNA synthesis is equal to the predicted theoretical sum. No other differences in WRT cell sensitivity to either IGF-I or insulin, or IGF-I and TSH and insulin and TSH, can be noted. This finding is confirmed by the demonstration of specific and sensitive binding sites for EGF on WRT cells cultured in 5% calf serum; these binding sites are not present on WRT cells adapted to grow in 0.5% calf serum. Present data support the hypothesis that EGF and serum growth actions are mediated through the same analogous pathway, which is, however, different from those of TSH and/or IGF-I and/or insulin.     

Decreased mitogenic activity in hypercholesterolaemic plasma

The mitogenic activity on rat aortic smooth muscle cells of plasma from patients with hypercholesterolaemia was compared to that of plasma from a normocholesterolaemic control group and to that obtained after lowering plasma cholesterol levels in the hypercholesterolaemic groups with simvastatin. Mean plasma total and low density lipoprotein cholesterol and apolipoprotein B concentrations in the two groups were 8.62 +/- 0.62 mmol l-1, 7.03 +/- 0.56 mmol l-1 and 1.96 +/- 0.14 g l-1, respectively, and 5.05 +/- 0.20 mmol l-1, 3.42 +/- 0.18 mmol l-1 and 0.80 +/- 0.04 g l-1, respectively, all differences significant at P less than 0.001. During simvastatin treatment these parameters decreased to 5.70 +/- 0.35, 4.15 +/- 0.33 mmol l-1 and 1.33 +/- 0.08 g l-1, all changes being significantly (P less than 0.001) lower than before treatment. Hyperlipidaemic plasma in untreated patients had a significantly lower mitogenic activity than normolipidaemic control plasma as measured by autoradiographic labelling of DNA after 3H-thymidine incorporation, 25.7 +/- 1.8% vs. 32.0 +/- 1.9%, P less than 0.05. Lowering plasma cholesterol levels in the hyperlipidaemic patients 'normalized' the mitogenic activity to 32.3 +/- 1.6%, P less than 0.05. The reason for this unexpected finding is not known.     

Increased mitogenic activity of scleroderma serum: inhibitory effect of human recombinant interferon-gamma.

OBJECTIVES--To investigate the role of platelet activation in the development of systemic sclerosis and the role of interferon-gamma (IFN gamma) in the inhibition of mitogenic activity induced by whole blood serum of patients with systemic sclerosis. METHODS--The mitogenic activity of whole blood serum in the absence or presence of different concentrations of IFN gamma (a potent inhibitor of induced collagen synthesis in dermal fibroblasts) and platelet-poor plasma derived serum were tested on human dermal fibroblasts by measuring incorporation of [3H]thymidine. Platelet activation was determined by quantification of plasma beta-thromboglobulin (beta-TG) using a beta-TG radioimmunoassay kit. RESULTS--The mitogenic activity was significantly increased in whole blood serum and in platelet-poor plasma derived serum of the patients compared with controls. In contrast, no significant increase in beta-TG concentration was observed in scleroderma platelet-poor plasma compared with control. Recombinant human IFN gamma had a greater inhibitory effect on the mitogenic activity induced by whole blood serum of patients than on that produced with control sera, at any concentration of IFN gamma tested. CONCLUSIONS--Our results suggest that mitogenic activity observed in the plasma of sclerodermic patients could originate from cells other than platelets and could be involved in the development of fibrosis. The potent inhibitory effect of IFN gamma on this proliferative activity may account for the beneficial effect of this cytokine in the treatment of progressive systemic sclerosis.     

Contribution of insulin-like growth factor (IGF)-I and IGF-binding protein-3 to mitogenic activity in bovine mammary extracts and serum.

Peripubertal development of the mammary gland is probably mediated by locally produced growth factors acting in concert with circulating mitogens. Our objective was to investigate the effect of recombinant human insulin-like growth factor-binding protein-3 (rhIGFBP-3) or insulin-like growth factor-I (IGF-I) antibodies on the IGF-I-related mitogenic activity of bovine serum and of mammary tissue extracts in primary mammary epithelial cell cultures. Cells were obtained from prepubertal female calf mammary tissue and cultured in three-dimensional collagen gels. An aqueous mammary parenchymal tissue extract (pooled from 20 prepubertal heifers) or serum (pooled from 3 heifers) at a concentration of 5% was added to the medium containing either rhIGFBP-3 or monoclonal or polyclonal antibodies to human IGF-I. Cell proliferation was evaluated using [methyl-3H]thymidine incorporation as a measure of DNA synthesis. Addition of mammary extracts stimulated DNA synthesis 545% compared with basal medium. Addition of serum stimulated DNA synthesis by 28%. Mitogenic activity of serum and added IGF-I was abolished by addition of rhIGFBP-3 in equimolar concentrations with IGF-I. For mammary extracts, mitogenic activity was inhibited by 35%, 50%, and 82% by the addition of rhIGFBP-3 at, respectively, 1, 2 and 4 times the molar IGF-I concentration in the extract. Addition of rhIGFBP-3 to basal medium reduced DNA synthesis by 26%, whereas IGF-I antibodies had no consistent effect. These results indicate that circulating and mammary-synthesized IGF-I and IGFBPs probably play a critical role in prepubertal development of the bovine mammary gland.     

Cytoplasmic transfer of the mitogenic response to platelet-derived growth factor.

BALB/c 3T3 mouse cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA and divide. When cells are treated with PDGF and then fused to untreated cells, the resulting heterokaryons become competent to replicate their DNA. Cytoplasts derived from PDGF-treated cells are also able to transfer the growth response to untreated cells. After cytoplasmic transfer to another cell, the strength of the PDGF-induced mitogenic signal is attenuated by a factor roughly proportional to the increase in total cytoplasmic volume. When RNA synthesis is blocked during PDGF treatment, cells do not acquire the capacity to transfer the PDGF growth signal to untreated cells. By contrast, exposure to cycloheximide during PDGF treatment has no effect. These observations suggest that cytoplasmic transfer of the growth response to PDGF (competence) is mediated by a PDGF-induced stable RNA rather than by PDGF itself or a PDGF--receptor complex. The onset of DNA synthesis in PDGF--control heterokaryons occurs a minimum of 11 hr after cell fusion. Thus the substance that is transferred in these cell fusions is not directly involved in DNA synthesis; rather, it seems to trigger a sequence of events culminating in DNA synthesis.     

Cytosolic glucocorticoid receptors in the porcine lung during development and after hypophysectomy or thyroidectomy

The ontogeny of fetal lung glucocorticoid receptors and their regulation by the fetal pituitary, adrenal and thyroid gland during lung maturation were investigated. Sites with a specificity typical of glucocorticoid receptors were detectable in lung cytosol, with the order of potency of steroids being dexamethasone greater than cortisol greater than corticosterone greater than 11-deoxycorticosterone greater than progesterone greater than 17 alpha-hydroxyprogesterone greater than oestradiol-17 beta congruent to testosterone congruent to androstenedione congruent to oestrone. The binding affinity for [3H]dexamethasone was high (Kd = 0.23-0.60 nmol/l) and showed an age-related decrease during the perinatal period when cortisol levels were high. After charcoal treatment of the cytosol, however, a decrease in binding affinity was not as clearly evident. The Kd decreased following hypophysectomy of fetuses; thyroidectomy had no significant effect. The concentration of glucocorticoid receptors was high from day 82 to day 100 of gestation (1437 fmol/mg protein) and declined progressively to a lower value at term and following birth (660 fmol/mg protein). Hypophysectomy, but not thyroidectomy, prevented the age-related decline in receptor concentration. Lung glycogen content declined with fetal ageing in association with increases in plasma concentrations of cortisol and thyroxine and with changes in Kd and Bmax, but appeared to be more closely associated with concentrations of thyroxine. Hypophysectomy of fetuses decreased concentrations of both cortisol and thyroxine and prevented the depletion of lung glycogen content. Preliminary results from thyroidectomized fetuses showed decreases in plasma thyroxine and lung glycogen content compared with day-82 fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)