Search PubMed⌕ Search

Biomedical subjects

D R Clemmons

Publications and source records attributed to D R Clemmons.

At least 145 records · Page 8Linked to original sources
PubMed1992

Effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone on serum IGF-binding proteins in calorically restricted adults.

To determine the effects of exogenous insulin-like growth factor-I (IGF-I) and GH on IGF-binding proteins (IGFBP)-1, -2, and -3, six healthy nonobese adult volunteers underwent two 2-week periods of diet restriction (20 Cal/kg.day), and during the last 6 days of the first period received either IGF-I (12 micrograms/kg.h by iv infusion over 16 h) or GH (0.05 mg/kg.day by sc injection). During the second 2-week study period, the alternate hormone was given. IGFBP-1 and -2 concentrations were determined by specific RIA, and changes in IGFBP-3 were assessed by ligand blotting. Free IGF-I concentrations were measured by size-exclusion high pressure liquid chromatography, followed by RIA. Diet restriction alone did not affect either IGFBP-1 or -2 significantly. IGF-I treatment increased IGFBP-1 from 78 +/- 46 ng/mL (mean pretreatment) to 137 +/- 64 ng/mL (P less than 0.001; mean for the last 4 days of IGF-I). IGF-I also caused an increase in IGFBP-2 from 315 +/- 136 to 675 +/- 304 ng/mL (P less than 0.001). GH injections caused a modest decline in IGFBP-1 concentrations but had no effect on IGFBP-2 concentrations. By ligand blotting, both IGF-I and GH caused a modest increase in IGFBP-3 band intensity. In three subjects diet restriction alone caused a small decrease in IGFBP-3 hand intensity, and this was reversed by hormone treatment. Free IGF-I concentrations in serum were increased from 1.6% to 4.4% of the total IGF-I during IGF-I infusions. GH injections caused a smaller increase in free IGF-I concentrations. The results show significant increases in IGFBP-1 and -2 during IGF-I infusion. The change in IGFBP-3, while significant, is quantitatively less than that in experimental animals that have been given IGF-I while undergoing dietary restriction. The net effect of the changes in these three forms of IGFBPs is not sufficient to maintain a normal IGF-I-binding capacity in serum, because free IGF-I levels were increased disproportionately during the IGF-I infusions. Because hypoglycemia was noted in these subjects despite insulin suppression, these alterations in IGFBPs might have changed the tissue bioavailability of IGF-I and facilitated its hypoglycemic effects.

Carrier Proteins↗
PubMed1992

Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.

Adipose Tissue↗
PubMed1992

Similar distribution of insulin-like growth factor binding proteins-1, -2, -3 in human fetal tissues.

The actions of insulin-like growth factors (IGFs) are modulated by several specific binding proteins (IGF BPs). Since the anatomical distribution of IGF BPs is likely to dictate IGF bioavailability we used immunocytochemistry to localize IGF BP-1, -2, and -3 in early second trimester human fetal tissues. Primary antisera were directed against hIGF BP-1, bIGF BP-2 and hIGF BP-3 respectively, and showed less than 5% cross-reaction with heterologous IGF BP species. The distribution of immunopositive staining was similar for each IGF BP in many tissues being prominent in the epithelial lining of the gut, kidney and lung; in epidermis, adrenal cortex and pancreatic endocrine tissue; and in association with membranes of skeletal muscle fibres and cardiocytes. Unlike IGF BP-1 -2, IGF BP-3 was barely detectable in liver and absent from epiphyseal chondrocytes. Conversely, IGF BP-3 alone was visualized within neurons of the cerebral cortex. The co-distribution of IGF BPs in many human fetal tissues suggests that they may co-ordinately regulate IGF bioavailability in target tissues and modify IGF: receptor interactions.

Blotting, Western↗
PubMed1992

IGF binding proteins: regulation of cellular actions.

The recent purification and structural analysis of the six forms of insulin-like growth factor binding proteins has opened up the possibility of studying their mechanism of action at the cellular level. While a great deal of information has been published regarding control of secretion of these proteins by cells, their modulation of the target cell actions of the IGFs remains incompletely studied. Both IGFBP-1 and 3 have been shown to be bipotential regulators. Using one set of culture conditions, both proteins appear to inhibit IGF-mediated growth while if other conditions are used they appear to stimulate growth. IGFBP-2, 5 and 6 have been less well studied in this regard, although several studies showing growth inhibition with IGFBP-2 have been published. IGFBP-4 appears to be consistently inhibitory and no studies showing stimulation have been published to date. Recent research has focused on the reason for these bifunctional effects. Clearly the ability to adhere to cell surfaces appears to be one important property of these proteins that results in their ability to potentiate or inhibit growth. Likewise, phosphorylation of IGFBP-1 on serine residues appears to be a structurally modifying change which results in altered growth regulation. The future will no doubt uncover other post-translational modifications that result in modulation of biologic activity.

Animals↗
PubMed1992

Endocrine events prior to puberty in heifers: role of somatotropin, insulin-like growth factor-I and insulin-like growth factor binding proteins.

We have utilized active immunization against growth hormone releasing factor (GRF) to investigate relationships among somatotropin (ST), insulin-like growth factor-I (IGF-I), IGF binding proteins (IGFBP) and ovarian function in heifers. Active immunization against GRF (GRFi) has been demonstrated to abolish episodic release of ST and decrease serum concentrations of IGF-I. In initial experiments investigating onset of puberty, breeds of heifers differing in growth rate and reproductive traits (Angus, Charolais and Simmental) were immunized against GRF or served as controls (immunized against carrier protein, human serum albumin, HSAi). GRFi decreased rate of muscle and skeletal growth, but increased deposition of adipose tissue. In Angus and Charolais, but not Simmental heifers, GRFi at 6 mo of age significantly delayed onset of puberty beyond 18 mo of age. Retrospective analyses of serum IGF-I revealed that GRFi heifers reaching puberty at a normal age had greater pre-treatment (6 mo of age) IGF-I than GRFi heifers in which puberty was delayed. Collectively, these results strongly indicate that the bovine hypothalamic-hypophyseal-ovarian axis is particularly sensitive to changes in metabolism at or near 6 mo of age. Another series of experiments tested the hypothesis that lowering serum IGF-I via GRFi initially at 3 mo of age would increase the percentage of Angus and Simmental heifers not reaching puberty. Three mo old Angus and Simmental heifers were assigned to GRFi (n = 18), HSAi (n = 14) or received no treatment (controls, n = 16). HSAi and GRFi heifers were unilaterally ovariectomized (ULO) at 6 mo of age. As anticipated, GRFi at a younger age increased percentage of heifers not reaching puberty; over 75% of control and HSAi heifers reached puberty by 14 mo of age compared to 22% of GRFi heifers. Serum and follicular fluid (FFL; follicles < or = 4 mm) concentrations of IGF-I were suppressed by GRFi. Serum, but not FFL concentrations of IGF binding protein-2 (IGFBP-2) were greater in GRFi than in HSAi heifers. GRFi delayed puberty apparently by suppressing follicular growth because number of follicles < or = 7 mm was significantly lower in GRFi than in HSAi heifers. In conclusion, active immunization against GRF at 3 or 6 months of age delays puberty in beef heifers. Delayed puberty was preceded by suppression of follicular growth, and decreased concentrations of IGF-I in serum and follicular fluid.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗
PubMed1991

Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: effects on affinity for IGF-I.

The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs). We and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes. This observation prompted us to determine whether IGFBP-1 undergoes posttranslational modifications. Immunoprecipitation was used to show that Chinese hamster ovary cells (transfected with a human IGFBP-1 cDNA construct) and human hepatoma (HepG2) cells secrete 32P-labeled IGFBP-1 following incubation with [32P]orthophosphate. Phospho amino acid analysis of 32P-labeled IGFBP-1 revealed only phosphoserine residues. A method was developed that could separate nonphosphorylated IGFBP-1 from four or five phosphorylated isoforms. Using this technique we demonstrated that human amniotic fluid and human fetal serum contain a large proportion of nonphosphorylated IGFBP-1, as well as phosphorylated forms. In contrast, HepG2 cells and human decidual cells secrete predominantly the phosphorylated isoforms. These observations suggest that IGFBP-1 is secreted as a phosphoprotein and is subsequently dephosphorylated in vivo. Binding studies showed that the phosphorylated IGFBP-1 secreted by HepG2 cells has a 6-fold higher affinity for IGF-I than it does after dephosphorylation. We conclude that IGFBP-1 is phosphorylated and that this phosphorylation is a physiologically important posttranslational modification.

Animals↗
PubMed1991

Secretion and biological actions of insulin-like growth factor binding proteins in two human tumor-derived cell lines in vitro.

The insulin-like growth factors (IGFs) I and II are present in extracellular fluids associated with specific binding proteins (IGFBPs) that can modify their biologic actions. These studies were undertaken to determine which forms of IGFBP are secreted by endometrial carcinoma (HEC-1B) and breast carcinoma (MDA-231) cells, to characterize variables that control IGFBP secretion, and to study the effect of IGFBP-1 and IGFBP-2 on IGF-I stimulated cell proliferation. Secreted IGFBPs were identified by ligand blotting and IGFBP-1 was quantified using a specific radioimmunoassay (RIA). MDA-231 cell conditioned media (CM) contained four (43,000, 39,000, 30,000 and 24,000 Mr) forms of IGFBP, and HEC-1B cell CM contained three forms (39,000, 34,000 and 30,000 Mr). Immunoblotting showed that the 30,000 Mr form secreted by both cell types was IGFBP-1. Likewise the 34,000 Mr band in HEC-1B media reacted with IGFBP-2 antiserum and the 39,000 and 43,000 Mr bands reacted with IGFBP-3 antiserum. IGF-I stimulated the secretion of IGFBP-3 from both cell types and IGFBP-2 from HEC-1B cells but either decreased or caused no change in secretion of IGFBP-1 and a 24,000 Mr form. In contrast, insulin inhibited the secretion of IGFBP-1 but increased the secretion of the 24,000 Mr form. Compounds that elevate intracellular cAMP levels increased the secretion of IGFBP-3, IGFBP-1, and the 24,000 Mr form from both MDA-231 and HEC-1B cells. When sparse cultures of MDA-231 cells were used, addition of IGF-I caused a 24% increase in cell number after 48 hr. This mitogenic response was enhanced by the presence of recombinant human IGFBP-1 (45% increase in cell number, P less than 0.001). Bovine IGFBP-2 did not potentiate IGF-I stimulated cell proliferation. These findings show that two tumor cell lines secrete distinct forms of IGFBPs and that there is differential regulation of IGFBP secretion. At least one form secreted by both tumors may act as a positive autocrine modulator of IGF-I's growth stimulating actions.

Breast Neoplasms↗
PubMed1991

Nutrient balance and stage of lactation affect responses of insulin, insulin-like growth factors I and II, and insulin-like growth factor-binding protein 2 to somatotropin administration in dairy cows.

Six Holstein cows were used in a complete block design to examine effects of period of lactation and somatotropin (bST) administration on concentrations of insulin, insulin-like growth factors (IGF-I, IGF-II), and IGF-binding protein 2 (IGFBP-2). During late lactation, the dry period and the subsequent early lactation, cows received injections of NaHCO3 buffer for 5 d and bST for 7 d. Cows were in positive energy and protein balances during late lactation and the dry period and in slight negative balances during early lactation. Basal insulin concentrations were highest in late lactation (170 pmol/L), whereas bST concentrations were higher in early lactation (0.6 micrograms/L). Insulin was increased by bST in the dry period (255 pmol/L) and late lactation (149 pmol/L) but not in early lactation (14 pmol/L), probably because of greater availability of glucose during positive nutrient balance. Basal IGF-I was lowest in early lactation (63.6 micrograms/L) but was increased by bST during all periods. The IGF-I response to bST administration was lower during early lactation (74.1 micrograms/L) compared with late lactation (123.6 micrograms/L) and dry period (146.0 micrograms/L). The IGF-II concentrations were not affected by period of lactation of bST administration but IGF-II tended to be higher during bST administration when cows were dry. Concentration of IGFBP-2 was higher during early lactation when cows were in negative nutrient balance (479.5 micrograms/L) than during the dry period (289.2 micrograms/L) and was decreased with bST. These data support a role of insulin and IGF in regulation of milk production. Availability of nutrients may be involved in regulating these hormones, particularly during bST treatment.

Animal Nutritional Physiological Phenomena↗
PubMed1991

Nutritional regulation of IGF-I and IGF binding proteins.

The secretion of both IGF-I and IGF-II, peptides that are structurally related to insulin, is linked to nutrient intake. These growth factors and their binding proteins appear to be major links between nutrient intake and cellular anabolic responses. Understanding the mechanisms by which nutrients control IGF synthesis and regulate transport to target tissues should permit the formulation of strategies for treatment of catabolic disorders. Understanding the alterations of such processes in catabolic states should enable development of better methods for nutritional repletion and wiser uses of anabolic agents in the treatment of these conditions.

Animals↗
PubMed1991

Colocalization of insulin-like growth factor-binding protein with insulin-like growth factor I.

We report the localization of insulin-like growth factor I (IGF-I) and a 25-kDa form of insulin-like growth factor-binding protein (IGF-BP-1) in adult rat kidney. The antigens were localized using a rabbit anti-human IGF-I antibody, and a rabbit anti-human IGF-BP-1 antibody raised against human 25-kDa IGF-BP-1 purified from amniotic fluid. Immunohistochemistry by the avidin-biotin peroxidase conjugate technique showed that both peptides are located in the same nephron segments, in the same cell types. The most intense staining was in papillary collecting ducts. There was moderate staining also in cortical collecting ducts and medullary thick ascending limbs of Henle's loop. In collecting ducts the antigens were shown to be present in principal cells but not in intercalated cells. In distal convoluted tubules, cortical thick ascending limbs, and in structures presumptively identified as thin limbs of Henle's loops there was only modest staining. The macula densa, however, lacked immunoreactivity. Colocalization of IGF-I and IGF-BP-1 in the same cells supports the notion, derived from studies on cultured cells, that the actions of IGF-I may be modified by IGF-BPs that are present in the same location.

Animals↗
PubMed1991

Retinoid modulation of insulin-like growth factor-binding proteins and inhibition of breast carcinoma proliferation.

Retinoids induce cellular differentiation and inhibit cellular proliferation. Proliferation of human breast carcinoma cells in vitro is markedly inhibited by these compounds. On the other hand, insulin-like growth factors (IGFs) and their receptors seem to be involved in the growth of certain breast carcinoma cells by autocrine or paracrine effects. Since the effects of both IGF-I and IGF-II may be modulated by specific binding proteins (IGF-BPs) we examined the possibility that one mechanism by which retinoic acid may inhibit cancer growth is by an alteration in these BPs, thereby blocking IGF's growth effect. Retinoic acid (RA; 1 microM) completely blocked the effect of IGF-I (50 ng/ml) on enhancing proliferation of MCF-7 cells in culture. This effect of RA was not associated with any significant change in specific IGF-I-binding sites on these cells. RA induced a 3-fold increase in IGF-binding activity in conditioned medium, measured using a polyethylene glycol-immunoglobulin precipitation assay and a charcoal absorption assay. This increase was associated with the appearance of 42- and 46-kDa IGF-BPs on ligand blotting. The effect of RA on these IGF-BPs was time and concentration dependent. In contrast, during some experiments the 27- and 36-kDa BPs actually decreased. These findings support the hypothesis that RA may inhibit the growth of certain breast carcinoma cells by increasing the secretion of certain IGF-BPs, which could directly modulate the growth effect of IGFs.

Breast Neoplasms↗
PubMed1991

Failure of insulin-like growth factor-I (IGF-I) infusion to promote growth in protein-restricted rats despite normalization of serum IGF-I concentrations.

Dietary protein restriction in young rats induces GH resistance characterized by growth arrest and low serum insulin-like growth factor-I (IGF-I) concentrations. To determine whether the low serum IGF-I concentrations are responsible for the stunted growth, we infused 4-week-old protein-restricted rats with recombinant human IGF-I (300 micrograms/day) or rat GH (200 micrograms/100 g body wt/day) by osmotic minipump for 1 week. Despite the normalization of serum IGF-I concentrations by IGF-I infusion, carcass growth was not stimulated. In contrast, growth of the spleen and kidney was enhanced (+45% and +28%, respectively). Serum IGF-binding protein 3 (IGFBP-3), the principal carrier of IGF-I in the serum at this age, is decreased by 34% in protein-restricted animals and restored to normal by IGF-I infusion. Contrary to the selective effects of IGF-I on growth of protein-restricted rats, well nourished hypophysectomized rats infused with 150 micrograms/day recombinant human IGF-I showed a significant growth response, including carcass and organ growth and normalization of IGFBP-3 values. These responses indicate that our IGF-I preparation and mode of delivery were effective. We conclude that: 1) dietary protein restriction causes organ-specific resistance to the growth-promoting properties of exogenous IGF-I; 2) IGF-I mediates the stimulatory effects of growth hormone on IGFBP-3 synthesis; and 3) the absence of carcass growth in the presence of normal serum IGF-I concentrations during infusion of IGF-I suggests that the growth arrest that accompanies protein restriction is mediated in part by resistance to IGF-I.

Animals↗
PubMed1991

The direct in vitro effect of insulin-like growth factors (IGFs) on normal bovine mammary cell proliferation and production of IGF binding proteins.

Mammary epithelial cells isolated from pregnant, nonlactating heifers were grown in vitro using collagen substrates. Using these systems, the truncated form of insulin growth factor-1 (IGF-1) (des-3-IGF-1), IGF-1, and IGF-2 all stimulated a significant (0.5 to 1 fold) increase in cell proliferation (des-3-IGF-1 greater than IGF-1 greater than IGF-2). When grown in media containing serum plus IGF-1, normal bovine mammary cells also produced and secreted at least four species of IGF-binding protein (IGFBP) ranging from 21K to 48K (as demonstrated by ligand blot analysis). However, cells grown in serum free media secreted detectable quantities of only 2 major forms of IGFBP of 34K and 48K. Using immunoblot analysis, these proteins were identified as IGFBP-2 and IGFBP-3, respectively. Both proteins were inducible by the addition of IGF to the serum free media (relative potency; IGF-1 greater than des-3-IGF-1 greater than IGF-2). Using RIA analysis, bovine mammary cells cultured in the presence of IGF-1 produced 20-25 ng/ml IGFBP-2 compared to control cultures which secrete approximately 1.0 ng/ml. Cells exposed to des-3-IGF-1 produced 40-60% less IGFBP-2 whereas insulin and IGF-2 did not stimulate significant IGFBP-2 production. These data indicate that normal bovine mammary cells secret IGFBP-2 and IGFBP-3. This secretion is stimulated by IGF-1 and des-3-IGF-1 suggesting a mechanism for regulating local IGF activity.

Animals↗
PubMed1991

Insulin-like growth factor (IGF) binding to cell monolayers is directly modulated by the addition of IGF-binding proteins.

Insulin-like growth factor-I (IGF-I) binds to specific receptors and IGF-binding proteins (IGFBPs) that are present on cell surfaces. The analysis of [125I]IGF-I binding to human fibroblasts is complicated by IGFBPs on the cell surface and their release into the medium during the binding assay. This release alters the distribution of [125I]IGF-I between type I IGF receptors and both soluble as well as cell surface-associated IGFBPs. In the present study we have determined the effects of three different forms of IGFBPs on [125I]IGF-I binding to cell surface binding sites of human fetal fibroblasts (GM10 cells) and porcine smooth muscle cells. Human 29,000 mol wt (Mr; IGFBP-1), bovine 34,000 Mr (IGFBP-2), and bovine 46,000 Mr (IGFBP-3) forms of IGFBP were compared. Each of the three IGFBPs inhibited [125I]IGF-I binding to the cell surface of both cell types. This effect was due to increased binding of [125I]IGF-I by the IGFBPs in the assay buffer. At equimolar concentrations, IGFBP-3 was more effective than either IGFBP-1 or IGFBP-2 in blocking cell surface binding. The addition of increasing concentrations of unlabeled IGF-I in the presence of each IGFBP showed that either IGFBP-1 or IGFBP-3, but not IGFBP-2, resulted in a paradoxical increase in [125I]IGF-I binding to the cell surface. The paradoxical increase occurred in the presence of excess insulin, indicating that unsaturated type I IGF receptors are not required to demonstrate this phenomenon. In a physiological salt solution, the order of affinity of the IGFBPs for IGF-I was IGFBP-3 greater than IGFBP-1 greater than IGFBP-2. These differences in affinity appear to account for the differences in IGF-I competition for binding that are seen when each of the three proteins is added. Thus, IGFBPs have the potential to alter the partitioning of IGF-I between cell surface-associated IGFBPs, membrane receptors, and the IGFBPs in extracellular fluids. The various forms of IGFBP affect IGF cell surface binding differently, and therefore, each may have distinct effects on IGF target cell actions.

Animals↗
PubMed1991

Regulation of insulin-like growth factor (IGF) binding proteins in transgenic mice with altered expression of growth hormone and IGF-I.

The insulin-like growth factors (IGFs) are present in extracellular fluids bound to specific, high affinity IGF binding proteins (IGFBPs). IGFBPs are believed to mediate IGF transport to tissues and to modulate their actions on target cells. To determine whether IGF-I can modulate IGFBP concentrations in blood and to distinguish the effects of IGF-I from those of GH, we assessed serum IGFBP concentrations in four genotypically distinct groups of sibling transgenic (Tg) mice that differed in respect to their expression of IGF-I and GH. This unique physiological situation was created by crossing IGF-I Tg mice to GH-deficient, dwarf mice in whom somatotrophs were genetically ablated by the expression of a diphtheria toxin transgene in the somatotrophs. Because both Tg mouse lines are hemizygous for their respective transgene, progeny of the cross differ genotypically, according to whether or not they carry one or both transgenes, and phenotypically in regard to their relative expression of IGF-I and GH. GH-deficient mice showed a 15.7-fold decrease in serum IGF-I and a 5.5-fold decrease in serum IGFBP-3, but no change in a serum doublet band of 29,000 to 34,000 Mr, as assessed by ligand blotting. When IGF-I was expressed in the GH-deficient mice, serum levels of IGF-I and IGFBP-3 were 69% and 64% of those in normal sera, respectively. The 29,000 to 34,000 Mr doublet bands also increased. The ternary 150 kilodalton IGF-IGFBP complex, however, was not restored, presumably because IGF-I has no influence on the expression of the acid-labile subunit in this complex. In mice with IGF-I overexpression, serum IGFBP-3 was increased 2.1-fold and the sum of the 29,000 to 34,000 doublet bands was increased 2.9-fold. Immunoblotting showed that the changes in the 29,000 to 34,000 Mr forms observed by ligand blotting appeared to be predominantly due to changes in IGFBP-2. The results show that IGF-I can induce IGFBP-3 and IGFBP-2 independently of GH and that IGF-I is a major controller of these binding proteins.

Animals↗
PubMed1991

Regulation of insulin-like growth factor binding protein synthesis and secretion in a bovine epithelial cell line.

The Madin-Darby bovine kidney cell line was used to examine regulation of insulin-like growth factor binding protein (IGFBP) synthesis by epithelial cells. Ligand and immunoblot analysis of conditioned media indicated that IGFBP-2 was the predominant IGFBP secreted by untreated cells. Treatment with forskolin decreased secretion of IGFBP-2 by 75 +/- 3% and induced the appearance of IGFBP-3 and 24,000 Mr IGFBP. Although insulin alone did not induce the appearance of either band, in the presence of forskolin it increased the IGFBP-3 and 24,000 Mr bands 4.2 +/- 1.1 and 7.3 +/- 0.9-fold, respectively, above the values for forskolin treatment alone. Exposure to forskolin resulted in a 3-fold decrease in the abundance of IGFBP-2 messenger RNA (mRNA), and a 30-fold increase in IGFBP-3 mRNA. An additional 2- to 3-fold increase in IGFBP-3 mRNA was observed when cells were treated with insulin plus forskolin. Treatment with insulin plus forskolin increased cell number 2-fold, compared to small increases (26%) observed with forskolin treatment alone. Since treatment with IGF-I or -II did not result in similar responses to those of insulin, IGF analogs with differing affinities for IGFBP and IGF type I receptor were tested. B-chain IGF-I (decreased affinity for IGFBP) increased cell number and enhanced forskolin's effects on IGFBP-3 secretion and mRNA abundance to the same extent as insulin, whereas [Leu24,1-62]IGF-I (decreased affinity for the type I IGF receptor) did not. Therefore, activation of the type I IGF receptor was required to elicit increases in cell number and IGFBP synthesis and secretion, and the actions of IGF-I and II were likely blocked by binding to the large amounts of IGFBP-2 that were secreted. These results are in direct contrast to studies with human fibroblasts in which IGF-I and [Leu24,1-62]IGF-I stimulate IGFBP-3 secretion, whereas B-chain IGF-I has only a minimal effect. The ability to differentially regulate secretion of different forms of IGFBPs by epithelial cells and the finding that regulation is distinct from that of fibroblasts may have important implications for understanding mechanisms by which IGFs and IGFBPs interact to regulate epithelial cell growth.

Animals↗