Production of Insulin-like Growth Factors and Their Binding Proteins in Primary Cultures of Rat Liver Parenchymal and Nonparenchymal Cells

Regulation of the production of insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBPs), and their related proteins by various hormones was investigated in primary cultures of rat liver parenchymal and nonparenchymal cells.

Freshly isolated parenchymal cells contained mRNAs of IGF-I, IGF-II, IGFBP-1, IGFBP-4, growth hormone (GH) receptor, and the acid-labile subunit (ALS), which forms a ternary complex with IGF-I and IGFBP-3; however, parenchymal cells did not express the IGFBP-3 gene. In contrast, nonparenchymal cells contained IGFBP-3 mRNA exclusively, as we reported previously [Takenaka et al. Agric. Biol. Chem., 55, 1191–1193 (1991)]. Cultured rat parenchymal cells produced IGF-I, IGFBP-1, and IGFBP-4 prominently. In these cells, secretion of IGF-I and the content of IGF-I mRNA was greatly increased in the presence of GH in the medium. Insulin also increased the production of IGF-I. Secretion of IGFBP-l into the medium was enhanced by treatment with glucagon, dibutyrylcyclic AMP (Bu₂cAMP), and dexamethasone (Dex) and these enhancements with glucagon and Dex reflected the increase in its mRNA content. Insulin depressed the secretion of IGFBP-l. The content of IGFBP-4 in the parenchymal cells was increased by insulin, Bu₂cAMP, and triiodothyronine (T₃), thereby enhancing the production of IGFBP-4 and secretion into the medium. Cultured liver nonparenchymal cells of rats produced IGFBP-1, IGFBP-3, and IGFBP-4. Secretion of IGFBP-l was increased by Bu₂cAMP in the medium, that of IGFBP-3 by IGF-I, and that of IGFBP-4 by both IGF-I and Bu2cAMP. Regulation of the production of IGFBP-3 by IGF-I was demonstrated in these investigations.

These results suggest that GH increases production of IGF-I in the parenchymal cells and this IGF-I, in turn, increases the production of IGFBP-3 in nonparenchymal cells. As we found GH also increases ALS production in parenchymal cells, by these mechanisms, GH increases the formation of the ternary complex of IGF-I, IGFBP-3, and ALS. This study clearly demonstrates the interrelationship between parenchymal and nonparenchymal cells in the production of IGF-I and IGFBPs in the liver.     

Effects of dexamethasone on the production of insulin-like growth factor-I and insulin-like growth factor binding proteins in primary cultures of rat hepatocytes.

The effects of dexamethasone (Dex) on insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 production were investigated in primary cultures of rat hepatocytes. Dex enhanced the secretion of IGFBP-1 as measured by ligand blot analysis but did not show any prominent effect on immunoreactive IGF-I secretion. EC50 of Dex on IGFBP-1 secretion was calculated to be 3 x 10(-8) M. The content of IGFBP-1 mRNA in the cells increased greatly in the presence of Dex but the IGF-I mRNA content did not change significantly under the same conditions. Insulin showed the opposite effect of Dex by decreasing the production of IGFBP-1 and the cellular content of IGFBP-1 mRNA. This effect of insulin was observed also with Dex in the medium. These results show that the gene expression of IGF-I and IGFBP-1 is differently regulated by glucocorticoids and insulin in primary cultures of rat hepatocytes. The results most possibly explain the in vivo effects of glucocorticoids and insulin in regulation of IGF-I and IGFBP-1 production by liver.     

Effects of Dexamethasone on the Production of Insulin-like Growth Factor-I and Insulin-like Growth Factor Binding Proteins in Primary Cultures of Rat Hepatocytes

The effects of dexamethasone (Dex) on insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-l production were investigated in primary cultures of rat hepatocytes. Dex enhanced the secretion of IGFBP-1 as measured by ligand blot analysis but did not show any prominent effect on immunoreactive IGF-I secretion. EC₅₀ of Dex on IGFBP-1 secretion was calculated to be 3 × 10^?8m. The content of IGFBP-1 mRNA in the cells increased greatly in the presence of Dex but the IGF-I mRNA content did not change significantly under the same conditions. Insulin showed the opposite effect of Dex by decreasing the production of IGFBP-1 and the cellular content of IGFBP-1 mRNA. This effect of insulin was observed also with Dex in the medium. These results show that the gene expression of IGF-I and IGFBP-1 is differently regulated by glucocorticoids and insulin in primary cultures of rat hepatocytes. The results most possibly explain the in vivo effects of glucocorticoids and insulin in regulation of IGF-I and IGFBP-1 production by liver.     

Biosensor measurement of the interaction kinetics between insulin-like growth factors and their binding proteins.

The binding kinetics of human insulin-like growth factor binding protein (IGFBP) 1-6 for recombinant human insulin-like growth factor (IGF) I and II were measured and compared in the present study using surface plasmon resonance biosensor technique. Different concentrations of IGFBPs (5-100 nM) were allowed to interact with the immobilized IGF-I or IGF-II on sensor chip surface. Both des(1-3)IGF-I and insulin are known to bind weakly to the IGFBPs and therefore are used as negative controls for the binding experiments. The resultant sensorgrams were analyzed by using simple 1:1 binding model to derive both the association rate (k(a)) and dissociation rate (k(d)) constants for IGFBP-IGF interactions. The k(a) values of IGFBPs are in the range of 1x10(4) to 9x10(5) M(-1) s(-1) for IGF-I and 7x10(3) to 1.7x10(6) M(-1) s(-1) for IGF-II, respectively. The orders of k(a) for both IGF-I and IGF-II are IGFBP-3>IGFBP-5>IGFBP-6>IGFBP-4>IGFBP-2>++ +IGFBP-1. The k(d) values of IGFBPs are in the range of 1.5x10(-5) to 2x10(-4) s(-1) for IGF-I and 3.6x10(-5) to 3.7x10(-4) s(-1) for IGF-II, respectively. The order of k(d) for IGF-I is IGFBP-6>IGFBP-5>IGFBP-4>IGFBP-3>IGFBP-2>++ +IGFBP-1 and that for IGF-II is IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-4>IGFBP-3>++ +IGFBP-1, respectively. The equilibrium affinity constants (K(A)) were calculated based on the ratio of k(a)/k(d) and were more precise than the published literature values based on competitive radioligand binding assays. The systematic study enables a direct comparison on the IGF-binding properties among the various IGFBPs, and the kinetic data provide additional information to delineate the physiological role of different IGFBPs in vivo.     

Regulation of insulin-like growth factor (IGF) binding protein-2 synthesis and degradation by platelet-derived growth factor and the IGFs is enhanced by serum deprivation in vascular smooth muscle cells

Growth factors such as platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-1) stimulate proliferation and migration of vascular smooth muscle cells (SMC). IGF-l bioactivity is modulated by high-affinity binding proteins (IGFBP) which are important regulators of these processes. Procine vascular SMC synthesize IGFBP-2 and IGFBP-4 in vitro. In the present study, levels of IGFBP-2 in conditioned media (CM) were increased approximately 1.6 to 2.2-fold when cells were exposed to PDGF (20 ng.ml) or insulin (5 μg/ml) for 24 hr following a 24 hr incubation in serum-free media, or following a 72 hr exposure to either growth factor. Similar increases in IGFBP-2 mRNA levels were observed. Exposure of cells to PDGF for 24 hr without prior serum deprivation resulted in smaller (47 ± 11%) increases in IGFBP-2 protein levels but failed to alter mRNA levels. IGF-1, FGF-b? and EGF failed to increase IGFBP-2 using either experimental paradigm. In contrast, IGFBP-2 protein levels were consistently decreased (75 ± 14%) after 72 hr of exposure to IGF-II without corresponding decreases in IGFBP-2 mRNA levels. Immunoprecipitation of [³⁵S] methionine-labeled IGFBP-2 indicated that this decrease was not due to a decrease in synthesis of IGFBP-2. Immunoblot analysis of CM from cells treated with IGF-II indicated that the decrease in intact protein corresponded with an increase in two non-IGF binding IGFBP-2 fragments of 22 and 14 kD. Increased abundance of these fragements was also observed following IGF-I exposure, although corresponding decreases in intact IGFBP-2 were not usually observed. The relative abundance of these fragments did not appear to be affected by treatment with PDGF or insulin. In contrast to IGFBP-2, regulation of the levels of IGFBP-4 in CM did not appear to be altered by serum deprivation. Insulin consistently increased IGFBP-4 mRNA and protein levels under all situations. PDGF tended to increase IGFBP-4 protein levels, although this effect was less consistent and not as great as the increase observe with insulin. Treatment with IGF-I or -ll consistently decreased IGFBP-4 levels in CM but tended to increase their mRNA levels under all situations. These data indicate that insulin, PDGF, and the IGFs regulate both IGFBP-2 and IGFBP-4. While PDGF and insulin stimulate IGFBP-2 and 4 synthesis, the IGFs appear to activate protease(s) which regulate IGFBP-2 and -4 levels post-translationally. The regulation of IGFBP-2 levels by each of these mechanisms appears to be amplified by serum deprivation, but this is not observed with IGFBP-4. © 1995 Wiley-Liss, Inc.     

Characterization of insulin-like growth factor binding proteins (IGFBPs) secreted by bovine adrenocortical cells in primary culture: regulation by insulin-like growth factors (IGFs) and adrenocorticotropin (ACTH).

In previous studies, we have shown that insulin-like growth factor II (IGF-II) stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I [1]. The steroidogenic effect of both IGFs is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we therefore characterized the IGFBPs secreted by bovine adrenocortical cells in primary culture, and investigated the effect of corticotropin (ACTH) and recombinant human IGF-I and IGF-II on the regulation of IGFBP synthesis. By Western ligand blotting, four different molecular forms of IGF-binding proteins were identified in conditioned medium of bovine adrenocortical cells with apparent molecular weights of 39-44 kDa, 34 kDA, 29-31 kDa, and 24 kDa. In accordance to their electrophoretic mobility, glycosylation status and binding affinity, these bands were identified by immunoprecipitation and immunoblotting as IGFBP-3, IGFBP-2, IGFBP-1, and deglycosilated IGFBP-4, respectively. Quantification of the specific bands by gamma counting revealed that, in unstimulated cells, IGFBP-3 accounts for approximately half of the detected IGFBP activity, followed by IGFBP-1, IGFBP-2 and IGFBP-4. ACTH treatment predominantly increased the abundance of IGFBP-1 and to a lesser extent IGFBP-3 in a time and dose-dependent fashion. In contrast, IGF-I or IGF-II (6.5 nM) preferentially induced the accumulation of IGFBP-3 (1.9-fold) and to a lesser extent of IGFBP-4, but did not show any effect on IGFBP-1. When ACTH and IGFs were combined, an additive stimulatory effect on the accumulation of IGFBP-3 and IGFBP-4 was observed. In contrast to their different steroidogenic potency, no significant difference in the stimulatory effect of IGF-I and IGF-II on IGFBP secretion was found. In conclusion, bovine adrenocortical cells synthesize IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4, and their secretion is regulated differentially by ACTH and IGFs. These results, together with earlier findings, suggest that IGF-binding proteins play a modulatory role in the regulation of differentiated adrenocortical functions. Therefore, bovine adult adrenocortical cells provide a useful tissue culture model in which the complex interactions between two IGF-ligands, at least four IGF binding proteins and two IGF-receptors can be evaluated.     

Expression and release of insulin-like growth factor binding proteins in isolated epiphyseal growth plate chondrocytes from the ovine fetus

Insulin-like growth factor-II (IGF-II) is an autocrine modulator of epiphyseal chondrogenesis in the fetus. The cellular availability of IGFs are influenced by the IGF-binding proteins (IGFBPs). In this study, we investigated the control of expression and release of IGFBPs from isolated epiphyseal growth plate chondrocytes from the ovine fetus by hormones and growth factors implicated in the chondrogenic process. Chondrocytes were isolated from the proliferative zone of the fetal ovine proximal tibial growth plate and maintained in monolayer culture at early passage number. Culture media conditioned by chondrocytes under basal conditions released IGFBPs of 24, 34, and 29 kDa, and a less abundant species of 39-43 kDa that were identified immunologically as IGFBP-4, IGFBP-2, IGFBP-5, and IGFBP-3, respectively. Messenger RNAs encoding each species were identified by Northern blot analysis within chondrocytes, as was mRNA encoding IGFBP-6. Exposure to IGF-I or IGF-II (13 or 26 nM) caused an increase in expression and release of IGFBP-3. The release of IGFBP-2 and IGFBP-5 were also potentiated without changes to steady state mRNA, and for IGFBP-5 this was due in part to a release from the cell membrane in the presence of IGF-II. Insulin (16.7 or 167 nM) selectively increased mRNA and the release of IGFBP-3, while cortisol (1 or 5 microM) inhibited both mRNA and release of IGFBP-2 and IGFBP-5. Transforming growth factor-beta1 (TGF-beta1) (0.1 or 0.2 nM) increased the expression and release of IGFBP-3, and caused an increase in mRNAs encoding IGFBP-2 and IGFBP-5. Neither growth hormone (GH), fibroblast growth factor-2, nor thyroxine (T(4)) had any effect on IGFBP expression or release. The results suggest that IGFBP expression and release within the developing growth plate can be modulated by IGF-II and other trophic factors, thus controlling IGF availability and action.     

Interaction of insulin,glucocorticoids, and protein kinase C in the regulation of insulin-like growth factor-binding protein-1 production by H4IIE rat hepatoma cells

A sensitive RIA was used to examine regulation of IGFBP-1 in H4IIE rat hepatoma cells. IGFBP-1 was stimulated up to tenfold by dexamethasone and corticosterone, and this stimulation was abolished by RU486. The effect of dexamethasone increased with time in culture. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 up to fourfold with a maximal effect in short-term culture. Dexamethasone and PMA were additive in stimulating IGFBP-1. Under basal conditions IGFBP-1 production was linearly related to cell density: however, stimulation by dexamethasone was greatest in confluent cells, and PMA had a greater effect in sparse cultures. Insulin inhibited IGFBP-1 up to 80%, and this effect diminished with time in culture but was unaffected by cell density. Dexamethasone was stimulatory in the presence of a maximal inhibitory concentration of insulin, and insulin was inhibitory in the presence of maximal dexamethasone from 3–48 h in culture, regardless of cell density. PMA abolished the inhibitory action of insulin on IGFBP-1 secretion and mRNA expression during incubation periods of less than 4 h and not during longer incubations. PMA did not influence the stability of IGFBP-1 mRNA. We conclude that, in rat H4IIE cells, dexamethasone and PMA stimulate IGFBP-1 by independent mechanisms and speculate that when protein kinase C is activated the inhibitory action of insulin is blocked. © 1996 Wiley-Liss, Inc.     

Production of insulin-like growth factor binding-proteins by bovine adrenomedullary cells: differential regulation by IGF-I and dexamethasone

In the present study we examined the production of insulin-like growth factor binding proteins (IGFBPs), in chromaffin cells, a model system for sympathetic neurons. Four IGFBPs of approximately 27, approximately 31, approximately 36 and a doublet of approximately 45-50 kDa, detected in Western ligand blots of conditioned medium, were identified in Western immunoblots as IGFBP-4, IGFBP-5, IGFBP-2 and IGFBP-3, respectively. In ligand blots IGFBP-3 and IGFBP-4 appeared as the most prominent species. IGF-I (1 nM) enhanced release of IGFBP-3 while dexamethasone (1 nM) diminished release of IGFBP-4. No significant proteolytic degradation of the IGFBPs was demonstrated. Cycloheximide completely attenuated release of the IGFBPs, indicating dependency on new synthesis of the proteins. These findings are consistent with autocrine modulation of the IGF system in bovine adrenomedullary chromaffin cells by IGFBPs. Furthermore, the specific stimulatory and inhibitory effects of IGF-I and dexamethasone, respectively, on release of the predominant species of IGFBP-3 and IGFBP-4, suggested that IGFBP production may be selectively modulated in a positive and negative manner.     

Factors regulating insulin-like growth factor binding protein-3 secretion from human hepatoma (HepG2) cells

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is a growth hormone (GH) dependent carrier of the IGFs in human serum. Apart from GH regulation the hormonal control of IGFBP-3 production is not well established and although the liver is considered to be the main source of circulating IGFBP-3, there are no in vitro studies of the effect of both insulin and IGFs on the IGFBP-3 produced in human hepatoma cells. The effect of sex hormones as well as cortisol has not been studied. To elucidate this we performed cell culture studies on HepG2 cells in the presence of various effectors. Insulin, IGF-I and IGF-II brought about a 1.5-2-fold enhancement of IGFBP-3 release at 7.5-30 nM concentrations. In contrast, cortisol decreased IGFBP-3 secretion by 30-40% whereas estradiol, tamoxifen and testosterone had no effect at physiological concentrations. We conclude that, in addition to GH, also insulin, IGF-I and IGF-II and glucocorticoids can modulate IGFBP-3 secretion by human hepatoma cells.     

Comparative studies on the regulation of insulin-like growth factor-binding protein-1 (IGFBP-1) and sex hormone-binding globulin (SHBG) production by insulin and insulin-like growth factors in human hepatoma cells

Production of insulin-like growth factor-binding protein-1 (IGFBP-1) by the liver is efficiently inhibited by insulin both in vivo and in vitro. Consequently, serum IGFBP-1 concentration reflects insulin bioactivity in portal vein. Sex hormone-binding globulin (SHBG) is another insulin-regulated liver-derived protein that has appeared promising in detecting individuals with portal hyperinsulinemia. We compared the regulation of IGFBP-1 and SHBG production by insulin and insulin-like growth factors (IGF-I and IGF-II) in human hepatoma cell cultures. Insulin equipotently inhibited IGFBP-1 and SHBG production, with maximal decrease in culture medium concentrations being about 35% for both proteins during 48 h of culture in serum-free medium. IGF-I and IGF-II also inhibited the IGFBP-1 and SHBG levels. We conclude that IGFBP-1 and SHBG are equally sensitive to ambient insulin concentrations in human hepatoma cell cultures, and the production of both proteins is also attenuated by the IGFs.     

Inhibition of Caco-2 cell proliferation by all-trans retinoic acid: role of insulin-like growth factor binding protein-6.

The present study examined the effects of all-trans retinoic acid (tRA) on proliferation and expression of the IGF system in Caco-2 human colon adenocarcinoma cells. tRA inhibited Caco-2 cell proliferation in a dose-dependent manner, with a 40 +/- 2% decrease in cell number observed 48 h after the addition of 1 microM tRA. Ligand blot analysis of IGFBPs in conditioned media revealed that Caco-2 cells produced three IGFBPs of M(r): 34,000 (IGFBP-2), 24,000 (IGFBP-4), and 32,000 (IGFBP-6). The concentrations of IGFBP-2 and IGFBP-4 decreased by 48 +/- 6 and 70 +/- 13%, respectively, whereas that of IGFBP-6 increased by 698 +/- 20% with 1 microM tRA. tRA decreased mRNA levels of IGFBP-2 and IGFBP-4 by 20 +/- 3 and 50 +/- 8%, respectively, whereas tRA increased IGFBP-6 mRNA by 660 +/- 20%. tRA did not alter levels of IGF-II mRNA or peptide. To examine if endogenous IGFBP-6 inhibits cell proliferation, Caco-2 cells were transfected with an IGFBP-6 cDNA expression construct or pcDNA3 vector only and stable clones were selected. Clones overexpressing IGFBP-6 grew more slowly than vector controls and achieved final densities 30-55% lower than those of vector controls. Accumulation of IGFBP-6 mRNA and concentrations of IGFBP-6 peptide in conditioned media were increased by 200-250 and 220-250%, respectively, in the IGFBP-6 clones compared with controls. Increased expression of IGFBP-6, which has a high binding affinity for IGF-II, following tRA treatment suggests that the decreased proliferation caused by tRA may result, at least in part, from IGFBP-6-mediated disruption of the IGF-II autocrine loop in these colon cancer cells.