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

1988 ◽  
Vol 118 (4) ◽  
pp. 513-520 ◽  
Author(s):  
C. A. Conover ◽  
P. Misra ◽  
R. L. Hintz ◽  
R. G. Rosenfeld
Keyword(s):  
Binding Sites ◽  
Human Fibroblast ◽  
Type I ◽  
Type Ii ◽  
Low Concentrations ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Differential Binding ◽  
Igf Receptor ◽  
Binding Curves

Abstract. 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 μg/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 μg/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.

Stimulation of DNA synthesis in chicken muscle satellite cells by insulin and insulin-like growth factors: evidence for exclusive mediation by a type-I insulin-like growth factor receptor

1991 ◽  
Vol 128 (1) ◽  
pp. 35-NP ◽  
Author(s):  
M. J. Duclos ◽  
R. S. Wilkie ◽  
C. Goddard
Keyword(s):  
Growth Factors ◽  
Satellite Cells ◽  
Type I ◽  
Type Ii ◽  
Muscle Satellite Cells ◽  
Chicken Muscle ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Igf Receptor ◽  
Insulin Like Growth Factors

ABSTRACT Insulin-like growth factors-I and -II (IGF-I and IGF-II) stimulate proliferation, differentiation, nutrient uptake and protein accretion in muscle cells. These effects are thought to be mediated through the type-I IGF receptor although a role for the type-II IGF receptor cannot be ruled out, since it has been found in most cells studied so far. Current evidence suggests that the chicken does not have a type-II IGF receptor and therefore provides a good model to study the function of IGF peptides. We have compared the effects of insulin and insulin-like growth factors on DNA synthesis with the binding of these peptides to receptors in primary chicken muscle satellite cells. Human IGF-I (hIGF-I), hIGF-II and porcine insulin increased thymidine incorporation into DNA by threefold in muscle satellite cells prepared from neonatal chickens. IGF-I and -II were almost equipotent, with half-maximum effective concentrations of 10 μg/l, and were 1000-fold more potent than insulin. A combination of maximum effective concentrations of all three peptides was not additive, suggesting that their effect was mediated by the same receptor. Receptor binding studies on satellite cells demonstrated the presence of specific IGF receptors. Human IGF-I inhibited the binding of 125I-labelled hIGF-I with a much higher potency than insulin, as usually observed for a type-I IGF receptor. However, unlabelled hIGF-II exhibited a higher potency than hIGF-I in displacing 125I-labelled hIGF-I. Affinity cross-linking of 125I-labelled hIGF-I and -II, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that hIGF-I and -II bound to a receptor with the structural characteristics of a type-I IGF receptor and confirmed the lack of a type-II IGF receptor in these cells. The concentrations of IGF-I, -II and insulin required for biological action and to displace 125I-labelled hIGF-I binding were similar, and support the hypothesis that their effects on proliferation were mediated exclusively through a type-I IGF receptor. Journal of Endocrinology (1991) 128, 35–42

Glucose regulation of the IGF response system in chondrocytes: induction of an IGF-I-resistant state

1999 ◽  
Vol 276 (4) ◽  
pp. R1164-R1171 ◽  
Author(s):  
K. M. Kelley ◽  
T. R. Johnson ◽  
J. Ilan ◽  
R. W. Moskowitz
Keyword(s):  
Glucose Concentration ◽  
Northern Analysis ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Receptor Mrna ◽  
Response System ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Igf Receptor

Nonresponsiveness to the growth-stimulatory actions of insulin-like growth factor (IGF)-I in chondrocytes has been reported in a number of disease states associated with impaired glucose metabolism. Primary rabbit chondrocytes were investigated for changes in their IGF response system [type-I IGF receptor and IGF-binding protein (IGFBP) expression] and in their ability to mount a synthetic response to IGF-I [as35S-labeled proteoglycan ([35S]PG) production] in media containing varying ambient glucose concentrations. Whereas basal [35S]PG synthetic rate was unaffected by glucose concentration, synthetic responsiveness to IGF-I was lost in media containing <5 mmol/l glucose or in media containing a “diabetic” glucose concentration (25 mmol/l). IGFBP expression, as measured by Northern analysis of mRNA levels and Western ligand blotting of secreted protein levels, was not significantly altered in the different glucose media, nor were there any differences in the cell surface localization of IGFBPs as assessed by affinity cross-linking with 125I-labeled IGF-I, suggesting that IGFBPs do not induce the IGF-I resistance. The nonresponsiveness to IGF-I in reduced glucose occurred with 25–50% reductions in steady-state levels of IGF type-I receptor mRNA and protein. A significant correlation between IGF receptor mRNA level and synthetic response to IGF-I was observed between 0 and 10 mmol/l glucose concentrations, suggesting that the loss of responsiveness in reduced glucose is manifested at the level of transcription and/or receptor mRNA stability. In contrast, nonresponsiveness to IGF-I in chondrocytes in diabetic glucose concentrations occurred without changes in receptor mRNA and protein levels, suggesting that IGF-I resistance was due to post-ligand-binding receptor defects. It is proposed that IGF-I resistance in chondrocytes subjected to inappropriate glucose levels may constitute an important pathogenic mechanism in degenerative cartilage disorders.

Immunoreactive and receptor-active insulin-like growth factor-I (IGF-I) and IGF-binding protein in blood plasma from the freshwater fish Macquaria ambigua (golden perch)

1993 ◽  
Vol 136 (2) ◽  
pp. 191-198 ◽  
Author(s):  
T. A. Anderson ◽  
L. R. Bennett ◽  
M. A. Conlon ◽  
P. C. Owens
Keyword(s):  
Binding Protein ◽  
Size Exclusion ◽  
Type I ◽  
Factor I ◽  
Type I Igf Receptor ◽  
Receptor Assay ◽  
Igf I ◽  
Igf Binding ◽  
Igf Receptor ◽  
Igf Binding Protein

ABSTRACT The presence of insulin-like growth factor-I (IGF-I)-related molecules and IGF-binding factors in blood from golden perch, Macquaria ambigua, an Australian native freshwater fish, was investigated. Serum was acidified to dissociate IGF and IGF-binding protein complexes that might be present, and fractionated by size-exclusion high-performance liquid chromatography at pH 2·8. Fractions were neutralized and their activities assessed by (i) an immunoassay for mammalian IGF-I which also detects chicken IGF-I but in which all known forms of IGF-II react very poorly, (ii) a receptor assay for IGF-II in which all known forms of IGF-I react poorly, and (iii) a type-I IGF receptor assay in which mammalian IGF-I and IGF-II polypeptides are almost equivalent. No IGF-II-like activity was detected. Three peaks of IGF-I-like activity were detected by IGF-I immunoassay and type-I IGF receptor assay. The major peak of activity was similar in molecular size to human IGF-binding protein-3, 45–55 kDa ('large IGF'), and a minor peak of activity which was similar in size to mammalian IGFs, 7·5 kDa. A third peak of activity was observed eluting at a time which indicates that it is a smaller molecule than any previously described IGF. The large IGF was temperature-sensitive, but was not a binding protein for 125I-labelled human IGF-I (hIGF-I). This material therefore was able to bind to anti-hIGF-I antibodies and to human type-I IGF receptors, and may represent the fish equivalent of mammalian prepro-IGFs. The two smallest forms of IGF activity identified by IGF-I radioimmunoassay and type-I radioreceptor assay following acidic size-exclusion chromatography were able to stimulate protein synthesis by L-6 myoblasts in culture, although large IGF did not. When fresh (but not frozen and thawed) golden perch serum was incubated with 125I-labelled hIGF-I and then fractionated by size-exclusion liquid chromatography at pH 7·4 through Sephadex G-100, the radioactivity became associated with a complex, intermediate in size between free IGF-I and the major IGF-binding protein in human serum. The association of 125I-labelled hIGF-I with the complex was inhibited by the presence of unlabelled hIGF-I in the incubation. These studies show that receptor-active, immunoreactive and bioactive IGF-I-like activity is present in golden perch serum, and demonstrate the presence of an IGF-I-binding factor in this species. Journal of Endocrinology (1993) 136, 191–198

scholarly journals Dexamethasone-induced growth inhibition of porcine growth plate chondrocytes is accompanied by changes in levels of IGF axis components

2002 ◽  
Vol 174 (2) ◽  
pp. 343-352 ◽  
Author(s):  
JJ Smink ◽  
JA Koedam ◽  
JG Koster ◽  
SC van Buul-Offers
Keyword(s):  
Growth Inhibition ◽  
Growth Plate ◽  
Growth Retardation ◽  
Culture Conditions ◽  
Type I ◽  
Mrna Levels ◽  
Growth Plate Chondrocytes ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Igf Receptor

High (pharmacological) doses of glucocorticoids inhibit the proliferation of growth plate chondrocytes, which leads to one of the side-effects of these steroids, namely suppression of longitudinal growth. Growth inhibition by glucocorticoids is thought to be mediated in part by impaired action of components of the IGF axis, which are important for chondrocyte regulation and hence for longitudinal growth. The aim of the present study was to determine whether glucocorticoid-induced growth retardation involves changes in IGF axis components. Chondrocytes were isolated from epiphyseal growth plates of neonatal piglets and treated with pharmacological doses of dexamethasone (DXM) for 24 h to study glucocorticoid-induced growth retardation. Under IGF-I-supplemented (10 nM) culture conditions, IGF-binding proteins (IGFBPs)-2, -4 and -5 were secreted by the growth plate chondrocytes and IGFBP-2 protein and mRNA levels were decreased by the DXM treatment, whereas IGFBP-4 and -5 were not affected. Proliferation of the chondrocytes, as measured by [(3)H]thymidine incorporation, was 3.5-fold higher in serum-supplemented medium in contrast to IGF-I-supplemented (10 nM) medium. In the presence of serum, DNA synthesis was significantly inhibited by 50-63% when treated with 100 nM DXM, which was prevented by the glucocorticoid-receptor antagonist Org34116. mRNA levels of IGF axis components were determined using Northern blot analysis. IGFBP-2 to -6 were expressed in the chondrocytes, IGFBP-1 was absent and both IGF-I and IGF-II, and the type I and type II IGF receptors were expressed. Treatment with DXM (100 nM) resulted in a 2-fold increase in mRNA levels of both IGFBP-5 and the type I IGF receptor, whereas IGFBP-2 mRNA levels decreased by 55%, in concert with the decrease in protein level observed under IGF-I-supplemented culture conditions. The changes in mRNA levels due to the DXM treatment were prevented by the glucocorticoid receptor antagonist. Our data show that exposure to pharmacological doses of DXM results in inhibition of proliferation and changes in components of the IGF axis, IGFBP-2 and -5 and the type I IGF receptor, suggesting a role for these components in glucocorticoid-induced growth retardation at the local level of the growth plate.

Characterization of type I IGF receptor and IGF-I mRNA expression in cultured human and bovine glomerular cells

1993 ◽  
Vol 48 (1-2) ◽  
pp. 9-20 ◽  
Author(s):  
Hiromi Ohashi ◽  
Kenneth M. Rosen ◽  
Fannie E. Smith ◽  
Lydia Villa-Komaroff ◽  
Ramesh C. Nayak ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Type I ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Igf Receptor

Interaction of ovine somatomedin-C/IGF-I and IGF-I with specific IGF-I receptors on cultured muscle-derived fibroblasts

1987 ◽  
Vol 116 (2) ◽  
pp. 186-192 ◽  
Author(s):  
M. V. Dodson ◽  
B. A. Mathison ◽  
K. L. Hossner
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Competitive Binding ◽  
Cross Linking ◽  
Type I ◽  
Somatomedin C ◽  
Receptor Complexes ◽  
Igf I ◽  
Relative Molecular Weight ◽  
Igf Receptor

Abstract. Binding of 125I-insulin-like growth factor-I and 125I-ovine somatomedin-C/IGF-I to monolayer cultures of muscle-derived ovine fibroblasts is described. Preliminary competitive binding experiments indicate that ovine fibroblasts possess independent cell surface receptors for IGF-I. Affinity of rIGF-II for IGF-I binding sites is minimal; rIGF-II binds to Type I IGF receptors at 1/1000 the strength of IGF-I. Insulin binds to the Type I IGF receptor at 1/100 the strength of IGF-I, whereas ovine somatomedin-C/IGF-I displays equivalent IGF-I binding as evidenced by overlapping competition of ovine somatomedin-C/IGF-I for 125IIGF-I binding sites. Results from disuccinimidyl suberate cross-linking of 125I-IGF-I to muscle-derived ovine fibroblasts in the presence of related polypeptides verified the competitive binding data. Under reducing conditions, 125I-IGF-I: receptor complexes migrated to a relative molecular weight of approximately 135 000 daltons. Specific 125I-IGF-I binding was completely inhibited by 10−8 mol/l IGF-I, 7.2 × 10−8 mol/l ovine somatomedin-C/IGF-I, and 10−6 mol/l insulin and partially inhibited by 7.2 × 10−9 mol/l ovine somatomedinC/IGF-I and 6.5 × 10−8 mol/l rIGF-II. 125I-ovine somatomedin-C/IGF-I: receptor complexes also migrated at a relative molecular weight of 135 000 daltons. No migratory band was observed at 250 000 to 260 000 daltons with either 125I-IGF-I or 125I-ovine somatomedin-C/IGF-I indicating that little labelled moiety bound to the Type II IGF receptor. Based on these preliminary competitive binding studies and cross-linking data, we conclude that ovine somatomedin-C/IGF-I is primarily interacting with the Type I IGF membrane receptor on ovine skeletal muscle fibroblasts.

Sign in / Sign up

Export Citation Format

Share Document