Biomedical subjects

C A Conover

Publications and source records attributed to C A Conover.

At least 91 records · Page 5Linked to original sources

PubMed1991

Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: evidence for processing of cell-bound IGFBP-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3E. coli) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3CHO) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3E. coli or hIGFBP-3CHO produced a dose-dependent inhibition of IGF-I but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGAFBP-3E. coli or hIGFBP-3CHO potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3E. coli for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3H]AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3E. coli with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 Mr hIGFBP-3E. coli seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3E. coli forms of 12,000-27,000 Mr. Cell-associated IGFBP-3E. coli (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3E. coli in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.

Aminoisobutyric Acids↗

PubMed1990

Regulation of insulin-like growth factor (IGF)-binding protein synthesis by insulin and IGF-I in cultured bovine fibroblasts.

Specific insulin-like growth factor-binding proteins (IGFBPs) are synthesized and secreted by bovine fibroblasts in vitro. By Western ligand blotting, three molecular forms of IGFBP were identified in conditioned medium from control cultures with mol wt (Mr) of 34,000, 28,000, and 24,000. Concentrations of these three IGFBP forms increased with time in serum-free conditioned medium without benefit of hormonal supplementation. Insulin and IGF-I were potent stimuli for IGFBP production by bovine fibroblasts, whereas bovine GH, epidermal growth factor, or steroid treatment had little or no effect. Insulin and IGF-I enhanced the production of 24,000, 28,000, and 34,000 Mr IGFBPs in a dose-dependent fashion. Moreover, addition of low nanomolar concentrations of insulin or IGF-I to bovine fibroblast cultures specifically induced the secretion of a 42,000/38,000 Mr species of IGFBP, which corresponded in size to the IGF-binding subunit of the principal 150,000 Mr IGFBP complex in serum. After stimulation with insulin or IGF-I, bovine fibroblasts (3 X 10(5) cells) secreted approximately 30 ng/24 h 42,000/38,000 Mr IGFBP. Subunits of 42,000/38,000 Mr in bovine fibroblast-conditioned medium did not form macromolecular complexes in either the absence or presence of bovine GH.

Animals↗

PubMed1990

Structural and biological characterization of bovine insulin-like growth factor binding protein-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) purified from bovine serum shares 19 of 25 amino-terminal amino acid residues with IGFBP-3 purified from human, rat, and porcine sources. A newly characterized bovine fibroblast model was used to investigate the biological effects of purified bovine IGFBP-3 (bIGFBP-3). Coincubation of insulin-like growth factor I (IGF-I) with increasing concentrations of bIGFBP-3 produced a dose-dependent inhibition of IGF-I-stimulated [3H]aminoisobutyric acid (AIB) uptake in cultured bovine fibroblasts. Inhibition was complete at equimolar concentrations of IGF-I and bIGFBP-3. Inhibition of IGF-I-stimulated [3H]AIB uptake paralleled the ability of bIGFBP-3 to prevent [125I]IGF-I cell surface binding. In contrast, preincubation with bIGFBP-3 resulted in a dose-dependent enhancement of IGF-I-stimulated [3H]AIB uptake; a 32-86% increase in IGF-I bioactivity was seen after a 24 h preexposure to 10 nM bIGFBP-3, and a 2- to 6-fold potentiation was seen after a 72 h preincubation. Preincubation with bIGFBP-3 increased both the sensitivity and maximal responsiveness of the cells to IGF-I. The potentiating effects of bIGFBP-3 were associated with increased [125I]IGF-I binding to cultured bovine fibroblasts. Affinity cross-linking experiments indicated that the increase in IGF-I binding was due to increased membrane-associated bIGFBP-3 rather than to a bIGFBP-3-induced increase in type I IGF receptors. bIGFBP-3 had no effect on insulin stimulation of [3H]AIB uptake under either experimental condition. These data suggest that soluble bIGFBP-3 inhibits IGF-I action by sequestering and preventing IGF-I receptor binding, whereas surface-associated bIGFBP-3 enhances the growth-promoting effects of IGF-I in bovine fibroblasts. We propose that IGFBP-3 serves a dual function in modulating IGF action in vivo.

Amino Acid Sequence↗

PubMed1990

Insulin regulation of insulin-like growth factor-binding protein production in cultured HepG2 cells.

A human hepatoma cell line, HepG2, secretes a discrete insulin-like growth factor-binding protein (IGFBP-1) into serum-free medium, which is identical to the 25K mol wt BP in amniotic fluid and plasma. IGFBP-1 levels in vivo have been shown to be inversely correlated with circulating insulin concentrations. This study investigated the direct effects of insulin on IGFBP-1 production in vitro. Addition of insulin to HepG2 cultures induced a rapid dose-dependent decrease in IGFBP-1 synthesis and secretion independent of the glucose concentration in the medium. As assessed by ligand binding and specific RIA, levels of IGFBP-1 were 20-50% of control levels in 18-h conditioned medium from insulin-treated cells. Monoclonal antibody studies indicated that the suppressive effect of insulin on IGFBP-1 synthesis was mediated through specific interaction with the insulin receptor. Therefore, HepG2 cells respond to insulin by altering the synthesis and secretion of IGFBP-1 in a manner that mimics many of the changes in plasma IGFBP-1 levels observed in vivo and provide an in vitro model for studies of IGFBP-1 biosynthesis.

Antibodies↗

PubMed1990

Lack of growth hormone effect on insulin-associated suppression of insulinlike growth factor binding protein 1 in humans.

Insulinlike growth factor binding protein 1 (IGFBP-1) has been shown to modulate the metabolic and mitogenic actions of the growth hormone (GH)-dependent peptide insulinlike growth factor I. Previous studies showed that levels of IGFBP-1 are regulated by insulin. The relative role of GH in the regulation of IGFBP-1 levels is less well defined and was examined in our study with a contiguous two-part protocol. Overnight (part A) and pre- and post-morning meal (part B) blood samples were obtained from eight healthy adults during a constant infusion of saline (SAL) or 4 micrograms.kg-1.min-1 GH. Five of eight subjects were restudied with glucose (GLUC) infused during part B (SAL + GLUC) to match glucose and insulin to levels observed during GH infusion. During SAL infusion, IGFBP-1 levels measured by specific radioimmunoassay showed a marked immediate decline after the evening meal in part A, with a subsequent nocturnal rise of 2.4- to 17.3-fold. GH infusion resulted in a similar meal-induced fall in IGFBP-1 levels but led to a delayed nocturnal rise in IGFBP-1, which was associated with elevated postprandial insulin concentrations. During part B, changes in plasma IGFBP-1 levels showed a similar pattern, with a delayed postprandial increase observed during both GH and SAL + GLUC infusions. The half-life of IGFBP-1 disappearance was calculated at approximately 2 h for all three infusion groups. Comparison of venous and arterialized blood samples showed no consistent pattern of difference, arguing against peripheral tissue clearance or compartmentalization as the mechanism for the rapid rise and fall in IGFBP-1 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1989

Serum glucocorticoids have persistent and controlling effects on insulinlike growth factor I action under serum-free assay conditions in cultured human fibroblasts.

The biological actions of insulinlike growth factor I (IGF-I) measured under serum-free assay conditions were found to be significantly influenced by prior subculture conditions for adult human fibroblasts. Glucocorticoids seemed to be the major medium variable affecting IGF-I action. IGF-I added to serum-free cultures had little or no effect on [14C]aminoisobutyric acid uptake or [3H]thymidine incorporation in human fibroblasts previously maintained in media containing serum with low glucocorticoid levels or in serum stripped of endogenous steroids. However, a 48-h preincubation with dexamethasone resulted in a marked synergistic increase in IGF-I stimulation of [14C]aminoisobutyric acid uptake and [3H]thymidine incorporation in these cultures. In contrast, IGF-I in serum-free medium seemed to be a potent mitogenic and metabolic stimulus for human fibroblasts which had been subcultured in media with a high glucocorticoid content, either endogenous or supplemented. After these culture conditions, a 48-h preexposure to dexamethasone had no further enhancing effect on IGF-I action. Dexamethasone also potentiated IGF-I, insulin, and epidermal growth factor stimulation of fibroblast replication depending on the earlier subcultivation conditions. Thus, glucocorticoids are important modulators of IGF-I bioactivity in cultured human fibroblasts. Serum glucocorticoids can exert a profound influence on the biological phenomena measured in cell culture, even when the serum has been removed before the actual experiment, and must be carefully taken into account for accurate evaluation of the biological function of IGF-I and other growth factors.

Adult↗

PubMed1989

Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation.

Specific, high affinity insulin-like growth factor (IGF) binding proteins are secreted by human fibroblasts in culture. By multiple criteria, the species of IGF binding proteins produced by human fibroblasts are distinct from the HepG2/amniotic fluid IGF binding protein, but share many characteristics with the growth hormone-dependent IGF binding protein forms predominant in normal adult human plasma. Treatment of cultured human fibroblasts with growth hormone produced an increase in IGF binding protein activity in the medium, while addition of glucocorticoids markedly diminished IGF binding activity. Insulin, epidermal growth factor, platelet-derived growth factor, and progesterone had no effect on IGF binding activity in fibroblast media. In comparison, HepG2 IGF binding activity was enhanced by progesterone, decreased by insulin, and unaffected by growth hormone or glucocorticoid treatment. Five molecular forms of IGF binding proteins were identified by Western ligand blots in human fibroblast conditioned medium, with Mr = 41,500, 37,000, 32,000, 28,000, and 23,000. In human fibroblast conditioned medium, the Mr = 41,500 and 37,000 IGF binding protein species were abundant, as in normal human plasma, with a major Mr = 23,000 form which was a minor component in plasma.

Animals↗

PubMed1989

Insulin-like growth factor receptors and binding protein in rat neuroblastoma cells.

B104, an established rat neuroblastoma cell line exhibiting specific neuronal qualities, was chosen as a model to study insulin-like growth factor (IGF) binding and action in the central nervous system. Specific binding of [125I]IGF-II to B104 membranes averaged 12.2 +/- 4.0% (mean +/- SD)/100 micrograms/ml protein compared with [125I]IGF-I binding of 10.1 +/- 2.9%. In competitive binding studies employing [125I]IGF-II as the radioligand, high affinity for IGF-II was demonstrated (50% displacement at 2.7 ng/ml), with none for IGF-I or insulin. Upon cross-linking [125I]IGF-I to membranes under reducing conditions, two prominent bands were observed, migrating with apparent mol wt (Mr) of 135,000 and 280,000. Both bands were inhibited by IGFs and insulin, but not by R-II-PABI, a polyclonal antibody to the type 2 receptor. These bands presumably represent the alpha-subunit and an incompletely reduced alpha-alpha-dimer of the type 1 IGF receptor. When cross-linking [125I]IGF-II to membranes under reducing conditions, the primary labeled bands migrated with apparent Mr of 260,000 and 280,000. These bands were inhibited by IGF-II and R-II-PABI, but not by insulin, and probably represent the monomeric type 2 receptor. In addition, we observed a minor band at apparent Mr 35,000, which was inhibited by IGF but not by insulin. By a modified cross-linking technique, we confirmed the existence of a small IGF-binding protein in the serum-free conditioned medium of B104 cultures, migrating as two bands with apparent Mr of 33,000-39,000. These proteins demonstrated high affinity for IGF-I and IGF-II, but none for insulin. In summary, this study demonstrates the presence in B104 rat neuroblastoma cells of 1) abundant classical type 1 and type 2 IGF receptors, and 2) a secreted and membrane-associated small IGF-binding protein.

Animals↗

PubMed1989

Structural and immunological comparison of insulin-like growth factor binding proteins of cerebrospinal and amniotic fluids.

The insulin-like growth factors (IGFs) found in plasma and a variety of other body fluids are complexed to specific binding proteins (BPs). The cDNA for a 25K IGF-BP was recently cloned and sequenced, and the primary structure of the BP deduced. This BP is found in amniotic fluid, decidual tissues, conditioned medium from HepG2 human hepatoma cells, and fetal plasma. An additional small IGF-BP has been identified in human cerebrospinal fluid (CSF). We now demonstrate that the IGF-BP found in CSF is structurally and immunologically distinct from that found in HepG2 conditioned medium. While the latter BP has approximately equal affinities for IGF-I and -II, the CSF BP has a 10- to 20-fold greater affinity for IGF-II. In affinity cross-linking studies under reducing conditions, the CSF BP had an apparent mol wt (Mr) of 32,000, while the HepG2 BP migrated as a doublet, with apparent Mr values of 30,000 and 28,000. On Western ligand blots, CSF BPs migrate as five discrete bands, with the most prominent band at an apparent Mr of 34,000, while HepG2 medium yielded a single band at an apparent Mr of 30,000. A polyclonal antibody developed against the human amniotic fluid BP immunoprecipitated the HepG2 BP and reacted with both the HepG2 and amniotic fluid BPs on Western blots, but failed to react with the CSF BP. These data indicate that the CSF and amniotic fluid/HepG2 BP are structurally and immunologically distinct small IGF-BPs.

Amniotic Fluid↗

PubMed1989

Cultured human fibroblasts secrete insulin-like growth factor IA prohormone.

Nucleotide sequencing of the two known cDNAs encoding human insulin-like growth factor I (IGF-I) predicts the existence of two different prohormone forms of IGF-I. The E peptide regions extend the carboxy-terminus of IGF-I by either an additional 35 (IGF-IA) or 77 (IGF-IB) amino acids. With a specific and sensitive RIA employing an antiserum directed against a synthetic peptide that is unique to the E peptide region of IGF-IA prohormone (EIA), we have identified EIA-immunoreactive material in the conditioned medium of fetal and postnatal human fibroblasts in culture. Incubation of postnatal human fibroblasts with GH increased specific immunoreactive EIA secretion 2- to 3-fold. There was no immunologically detectable 7.5K IGF-I or IGF-II peptide in acid-chromatographed human fibroblast-conditioned medium under either basal or GH-stimulated conditions. Acid chromatography of human fibroblast-conditioned medium on Sephadex G-75 revealed a single elution peak of EIA immunoreactivity corresponding to a mol wt of 9-17 K. With neutral chromatography, EIA immunoreactivity eluted at 25-38K mol wt. These data suggest that the E peptide region of IGF-IA is translated and released as part of the prohormone form in cultured human fibroblasts, and that the levels of this prohormone are regulated by GH.

Amino Acid Sequence↗

PubMed1988

Differential binding of 125I-IGF-I preparations to human fibroblast monolayers.

Specific, high affinity binding of 125I-IGF-I to the type I IGF receptor on human fibroblast monolayers was not altered by varying feeding schedules, serum lots, washing procedures, or incubation times and temperatures. However, markedly different competitive binding curves were obtained when different iodinated IGF-I preparations were used. Five of six radioligands bound preferentially to the type I IGF receptor on human fibroblast monolayers, with 50% displacement at 4-8 micrograms/l unlabelled IGF-I; with one radioligand a paradoxical 20-200% increase in 125I-IGF-I binding was observed at low concentrations of unlabelled IGF-I, while concentrations as high as 100 micrograms/l IGF-I failed to displace this radioligand. The latter binding pattern cannot be accounted for by 125I-IGF-I binding to the type II IGF receptor. These data indicate that various radioligands may have preferential affinities for different IGF-I binding sites on human fibroblast monolayers.

Autoradiography↗

PubMed1987

Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-II receptor: comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I.

Although insulin-like growth factors (IGF) I and II bind with high affinity to structurally discrete receptors, they bind with a lesser affinity to each other's receptor. We have evaluated the affinity of five different IGF-I preparations (three natural IGF-I preparations, one synthetic preparation, and one recombinant DNA-derived) for the IGF-II receptor in rat placental membranes, 18-54,SF cells and BRL-3A cells. In all tissues tested, the natural IGF-I preparations demonstrated an affinity for the IGF-II receptor which was 10-20% that of IGF-II. However, the recombinant and synthetic IGF-I preparations exhibited substantially lower affinities than natural IGF-I for this receptor, with only 10-25% reduction in (125-I)iodo IGF-II binding at peptide concentrations up to 400 ng/ml. Radioimmunoassay of the natural IGF-I preparations with an antibody directed against the unique C-peptide region of IGF-II demonstrated that contamination of IGF-I preparations with immunoreactive IGF-II could not exceed 5%. These results demonstrate that IGF-I purified from human plasma has a different affinity for the IGF-II receptor than does synthetic or recombinant IGF-I. Furthermore, these data are consistent with the hypothesis that IGF-I, itself, may be heterogeneous, and that subforms may vary in their affinities for the IGF receptors. Alternatively, IGF-I preparations which have been considered to be pure may be contaminated with small amounts of IGF-II, resulting in overestimation of the affinity of IGF-I for the type II IGF receptor.

Adult↗

PubMed1987

Insulin-like growth factor II binding and action in human fetal fibroblasts.

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.

Aminoisobutyric Acids↗

PubMed1987

Somatomedin C/insulin-like growth factor I binding and action in human fibroblasts aged in culture: impaired synergism with dexamethasone.

Somatomedin C/insulin-like growth factor I (SM-C/IGF-I) binding and action were compared in human fibroblasts serially passaged in vitro. Binding of [125I]SM-C/IGF-I was identical in early and late passage cells, with 50% displacement at 4 ng/ml SM-C/IGF-I. Maximal concentrations of SM-C/IGF-I (100 ng/ml) stimulated [3H]thymidine incorporation equally in both early and late passage cells. Furthermore, dose response curves indicated that there was no alteration in the sensitivity of cells to SM-C/IGF-I with age. Preincubation with dexamethasone (10(-7) M) potentiated SM-C/IGF-I's mitogenic effect in early passage cells. In contrast, dexamethasone had no effect on SM-C/IGF-I stimulation of [3H]thymidine incorporation or replication of late passage cells. These data indicate that fibroblasts passaged in vitro have normal SM-C/IGF-I binding and are capable of responding to the mitogenic effect of SM-C/IGF-I. The synergism between SM-C/IGF-I and dexamethasone in stimulating DNA synthesis and replication of early passage fibroblasts is lost, however, in late passage cells.

Cell Division↗

PubMed1987

Impaired synergism between somatomedin C/insulin-like growth factor I and dexamethasone in the growth of fibroblasts from a patient with insulin resistance.

To explore a possible mechanism for the diminished growth potential in a patient with an unusual form of insulin resistance, somatomedin C/insulin-like growth factor I (SM-C/IGF-I) and insulin stimulation of [3H]thymidine incorporation and cell replication were compared in skin fibroblasts from the patient (DF) and normal controls. There appeared to be no generalized abnormality in cellular responsiveness to growth factors. In both DF and control cells, SM-C/IGF-I (50 ng/ml), insulin (100 ng/ml), and epidermal growth factor (5 ng/ml) stimulated [3H]thymidine incorporation 5-, 2-, and 6-fold, respectively. Low concentrations of human hypopituitary serum (0.25%) enhanced the effectiveness of SM-C/IGF-I and insulin to a similar extent in both DF and control cells. On the other hand, 10% calf serum stimulated [3H]thymidine incorporation 37-fold in control cells, while DF cells were only 50% as responsive. Preincubation of control cells with dexamethasone (10(-7) M) caused a marked synergistic increase in SM-C/IGF-I stimulated [3H]thymidine incorporation (15- to 20-fold in serum-free medium; 50- to 80-fold in 0.25% human hypopituitary serum). In contrast, preexposure to dexamethasone did not augment SM-C/IGF-I stimulation of thymidine incorporation into DNA of DF cells. Furthermore, the stimulation of cell replication by SM-C/IGF-I and insulin was potentiated by dexamethasone in control but not DF cultures. These data suggest that impairment of the synergistic action of glucocorticoids with SM-C/IGF-I and insulin regulation of fibroblast growth may be involved in the pathology of this insulin-resistant growth disorder.

Cell Division↗

PubMed1986

Effect of an anti-insulin-like growth factor I receptor antibody on insulin-like growth factor II stimulation of DNA synthesis in human fibroblasts.

To investigate the role of insulin-like growth factor II in the control of DNA synthesis in human fibroblasts, dose-response curves for insulin-like growth factor I and II stimulation of [3H]thymidine incorporation were compared in the absence and presence of alpha IR-3, a highly specific monoclonal antibody directed against the type I insulin-like growth factor receptor. Specific binding of [125I]insulin-like growth factor I to human fibroblast monolayer cultures was inhibited 60-70% in the presence of alpha IR-3. alpha IR-3 had no effect on [125I]insulin-like growth factor II binding to human fibroblasts. However, alpha IR-3 inhibited both insulin-like growth factor I and II stimulated [3H]thymidine incorporation. These data indicate that the type II insulin-like growth factor receptor does not function as a transducer of insulin-like growth factor II's mitogenic effect in human fibroblasts.

Adult↗

PubMed1986

Hormonal control of the replication of human fetal fibroblasts: role of somatomedin C/insulin-like growth factor I.

Sparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin-like growth factor I (SM-C/IGF-I). At both developmental stages, the addition of SM-C/IGF-I (100 ng/ml) increased cell number at day 3 1.4-fold in serum-free medium and 2-fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose-response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth-promoting effects of SM-C/IGF-I, with a half-maximal response occurring at 6 ng/ml SM-C/IGF-I. This biological action of SM-C/IGF-I correlated with SM-C/IGF-I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM-C/IGF-I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM-C/IGF-I in stimulating replication of postnatal fibroblasts. The combination of SM-C/IGF-I (100 ng/ml), dexamethasone (10(-7) M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM-C/IGF-I and dexamethasone in fetal fibroblasts. In fetal cells, SM-C/IGF-I + EGF + PDGF +/- dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50-100% more responsive than postnatal cells to the proliferative effect of serum.

Blood Physiological Phenomena↗

PubMed1985

Comparative effects of somatomedin C and insulin on the metabolism and growth of cultured human fibroblasts.

At concentrations of 25 ng/ml in serum-free medium, somatomedin C (SM-C) and insulin stimulated 3H-thymidine incorporation in adult human fibroblasts 4- and 1.5-fold, respectively. The presence of 0.25% human hypopituitary serum (HHS), which by itself had little effect, enhanced the mitogenicity of both SM-C and insulin. Furthermore, 10(-7)M dexamethasone dramatically potentiated SM-C stimulation (70-fold) and insulin stimulation (28-fold) of 3H-thymidine incorporation. With dexamethasone and 0.25% HHS, significant stimulation of DNA synthesis was seen at 2.5 ng/ml for both SM-C and insulin. The effects of SM-C and insulin on 3H-thymidine incorporation were additive. These 3H-thymidine incorporation results were clearly supported by cell replication studies. On the other hand, SM-C and insulin had equivalent, nonadditive effects on RNA and protein synthesis and protein degradation. Half-maximal effects were seen for both peptides on all three metabolic processes at 2-5 ng/ml. In contrast to their synergism with SM-C in the stimulation of DNA synthesis and cell replication, HHS and dexamethasone did not enhance SM-C stimulation of RNA or protein synthesis or protein degradation. These data indicate that SM-C and insulin stimulate DNA, RNA, and protein synthesis, protein degradation, and cell replication in adult human fibroblasts at nanomolar concentrations, suggesting that each peptide is capable of acting through its own receptor. Both SM-C and insulin are also capable of synergism with low concentrations of serum and dexamethasone in the stimulation of DNA synthesis and cell replication. It is proposed that SM-C and insulin both participate in the regulation of cell growth and metabolism in vivo.

Adult↗