Rat Insulin-like Growth Factor Binding Protein-1 Research Articles

Physiological differences in insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation in IGFBP-1 transgenic mice.

Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulin-like growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six- to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [32P]H3PO4) secrete 32P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo.

Amino acid regulation of gene transcription of rat insulin-like growth factor-binding protein-1

To investigate the molecular mechanisms of increased transcription of the insulin-like growth factor-binding protein-1 (IGFBP-1) gene in dietary protein-deprived animals, the cis-acting sequence that is involved in this regulation was analyzed. We first showed that IGFBP-1 gene transcription was up-regulated by amino acid deprivation in cultured liver cell lines: H4IIE and HuH-7. Since HuH-7 cells showed a greater increase in IGFBP-1 mRNA in response to amino acid deprivation, this cell line was used in further experiments. Using a promoter function assay, we found that up-regulation of promoter activity responding to amino acid deprivation was abolished by deleting the region between -112 and -81 bp from the cap site from the gene construct. This cis-acting region includes the insulin-responsive element (IRE) and glucocorticoid responsive element (GRE) of IGFBP-1. In summary, the present observation suggests that the 32-bp (-112 to -81) in the IGFBP-1 gene 5' promoter region is involved in the induction of the IGFBP-1 gene in response to amino acid deprivation.

Factors regulating the rat insulin-like growth factor-binding protein-1 response to octreotide.

Insulin-like growth factor-binding protein-1 (IGFBP-1) production is increased by somatostatin and its analogues. In order to determine the time course and identify possible mechanisms of this increase in vivo we administered octreotide to rats and determined IGFBP-1 concentrations by RIA. After 60 min of anaesthesia, the mean baseline IGFBP-1 concentrations were 166 (95% confidence interval 123 to 225) ng/ml and increased in saline-infused animals to 729 (488 to 1086) ng/ml after 180 min. IGFBP-1 was stimulated transiently in response to octreotide, with circulating IGFBP-1 concentrations peaking at 1605 (1220 to 2111) ng/ml at 105 min during a continuous infusion of octreotide (100 micrograms/kg per h). In conscious chronically cannulated rats, baseline IGFBP-1 concentrations were 22 (18 to 28) ng/ml, 8-fold less than in the anaesthetised state, and were stimulated in the short term after administration of an octreotide bolus (100 micrograms/kg s.c.) to reach 88 (62 to 126) ng/ml at 60 min. A similar response was seen after i.v. administration to conscious rats. Intravenous bolus of octreotide (100 micrograms/kg) in rats anaesthetised for 3 h resulted in an increase in IGFBP-1 to peak at 1556 (1268 to 1910) ng/ml at 60 min. The IGFBP-1 response to octreotide was diminished in high-fat fed hyperinsulinaemic rats. The pattern of disappearance of iodinated IGFBP-1 from the circulation was not influenced by octreotide. The changes in GH, insulin and glucose concentrations alone did not sufficiently account for the patterns of response observed. We conclude that, in rats, octreotide stimulates IGFBP-1 acutely and this response is potentiated by factors related to anaesthesia.

Dexamethasone stimulation of rat insulin-like growth factor binding protein-1 promoter activity involves multiple cis-elements.

Insulin-like growth factor binding protein-1 (IGFBP-1) modulates the mitogenic actions of IGF-I and IGF-II. Dexamethasone increases IGFBP-1 mRNA abundance and gene transcription in rat liver and in H4-II-E rat hepatoma cells. A glucocorticoid response element (GRE) located at nucleotide (nt) -91/-77 is required for dexamethasone to stimulate rat IGFBP-1 promoter activity in transient transfection assays in H4-II-E cells. Mutagenesis of nt -108/-99 (the M4 region of the insulin response element), however, decreased dexamethasone-stimulated promoter activity despite the presence of an intact GRE, suggesting that regulatory sites in addition to the GRE were required for optimal dexamethasone stimulation. To identify these sites, we introduced 5'-deletion and substitution mutations into rat IGFBP-1 promoter fragments coupled to a luciferase reporter gene and transfected these constructs into H4-II-E cells. Three sites are required for optimal basal promoter activity: a site (nt -62/-50) that binds the liver-enriched transcription factor, hepatocyte nuclear factor-1 (HNF-1), the M4 site, and a putative binding site for transcription factor AP-2 (nt -293/-286). The HNF-1 and M4 sites and an upstream site (nt -252/-236) are also involved in dexamethasone stimulation under some, but not all, circumstances. Mutation of either the HNF-1 site or the M4 site decreased dexamethasone stimulation by more than 80% in constructs whose 5'-end was at nt -92, -135, or -235 but not if the 5' -end was at nt -278 or -327. These results suggest that the nt -278/-236 region can compensate for the loss of the HNF-1 site or the M4 site but that the HNF-1 and M4 sites do not compensate for each other in constructs whose 5'-end was at nt -135 or -235, which lack the nt -278/-236 region. The site within the nt -278/-236 region was localized to nt -252/-236 by deoxyribonuclease I protection and transfection assays. Thus, several cis-elements in the rat IGFBP-1 promoter cooperate, in varying combinations, with the low-affinity GRE to allow optimal dexamethasone-stimulated promoter activity.

Growth hormone modulates insulin regulation of hepatic insulin-like growth factor binding protein-1 transcription.

Hepatic transcription of insulin-like growth factor-binding protein-1 (IGFBP-1) is enhanced in hypophysectomized (hypox) rats and can be rapidly down-regulated by GH administration. Here we examined the effect of insulin on IGFBP-1 messenger RNA abundance in hypox rats and the effects of insulin and GH on IGFBP-1/chloramphenicol acetyltransferase (CAT) reporter plasmids transiently transfected into isolated hepatocytes from pituitary-intact and hypox rats. Unlike GH, administration of insulin to hypox rats in doses of 10 or 50 micrograms/100 g BW had no effect on hepatic IGFBP-1 messenger RNA abundance. Insulin at 10(-7) M resulted in a 42.1 +/- 9.8% suppression of CAT activity in hepatocytes from pituitary-intact animals transfected with a CAT reporter plasmid containing 1671 bp of the 5'-flanking region of the rat IGFBP-1 gene. In the same assay, GH at a concentration of 2.3 x 10(-8) M significantly reduced CAT activity. In contrast, insulin had no effect on CAT activity in hepatocytes from hypox rats, whereas GH resulted in comparable suppression of CAT activity in hepatocytes from hypox rats and pituitary-intact rats, 13.6 +/- 2.3% vs. 18.2 +/- 3.2%. Deletional analysis and mobility shift assays were used to identify the GH-responsive regions in the IGFBP-1 gene. GH suppression of CAT activity was lost when the IGFBP-1 5'-flanking region was deleted down to -277 bp, whereas insulin suppression was retained for all but the smallest fragment of the IGFBP-1 gene. Mobility shift assays were used to compare nuclear extracts from sham-operated, hypox, and GH-treated hypox rats. When hepatic nuclear extracts from hypox rats were incubated with the -277 to -82 and the -556 to -368 bp fragments, retarded bands were apparent that were not present in the extracts from sham-operated rats. GH treatment of hypox rats 15 or 30 min before death completely normalized the retardation pattern seen with the -277 to -82 bp fragment, but did not affect the pattern seen with the -556 to -368 bp fragment. A 20-bp fragment corresponding to the previously identified insulin response element, -108 to -89 bp, was also analyzed. An additional retarded band, not seen with nuclear extracts from sham-operated rats, was apparent when nuclear extracts of hypox rats or GH-treated hypox rats were used. These data provide the first in vitro evidence that GH directly regulates transcription of IGFBP-1 expression. In addition, our findings suggest that GH modulates insulin regulation of IGFBP-1 transcription, possibly by altering the milieu of trans-acting factors that interact with both the insulin response element and distinct upstream sites.

Impaired estrogen action in the uterus of insulin-like growth factor binding protein-1 transgenic mice.

Insulin-like growth factor-I (IGF-I) has been implicated as autocrine/paracrine mediator of estrogen action in the rodent uterus. Here, we examined the effects of 17-beta estradiol (E2), epidermal growth factor (EGF), and IGF-I on DNA synthesis in the uterus of ovariectomized (ovex) transgenic mice (Tg), which overexpress rat insulin-like growth factor binding protein-1 (IGFBP-1). Litter size was significantly reduced in Tg mice compared with wild-type mice. Immunohistochemical studies localized the expression of the transgene to the luminal and glandular epithelium. In addition, rat IGFBP-1 immunoreactivity was present in luminal secretions. E2-induced uterine DNA synthesis as measured by methyl-3H thymidine incorporation, was significantly reduced in ovex Tg mice; 4.77 +/- 0.59 and 4.97 +/- 0.53 for Tg strains 57C and 277A, respectively, compared with 8.65 +/- 0.73 fmol/microgram of DNA for Wt mice. Similarly, uterine weight after three daily injections of E2 was reduced in Tg mice compared with Wt mice; 2.85 +/- 0.39 vs. 4.23 +/- 0.26 mg/g BW, P < 0.01. Semiquantitative RT-PCR assays were used to demonstrate changes in uterine IGF-I messenger RNA (mRNA) and EGF mRNA abundance after administration of E2. An approximately 3-fold increase in IGF-I mRNA abundance was seen 6 h after E2 in both Tg and Wt mice. Over the same time course, little change was seen in EGF mRNA levels, which were similar in Tg and Wt mice. After 3 days of E2 treatment, an increase in EGF mRNA was apparent in Wt mice but not in Tg mice. The uterine DNA response to both IGF-I and EGF was significantly attenuated in Tg mice compared with Wt mice. The data reported here together with previous reports of E2 regulation of IGF-I and IGFBP-1 expression in uterus support the hypothesis that the IGF-I is a mediator of estrogen action in the uterus. In addition attenuation of the EGF response in the uterine tissue of Tg mice suggests that this response is also mediated, in part, by IGF-I.

Impaired estrogen action in the uterus of insulin-like growth factor binding protein-1 transgenic mice

Insulin-like growth factor-I (IGF-I) has been implicated as autocrine/paracrine mediator of estrogen action in the rodent uterus. Here, we examined the effects of 17-beta estradiol (E2), epidermal growth factor (EGF), and IGF-I on DNA synthesis in the uterus of ovariectomized (ovex) transgenic mice (Tg), which overexpress rat insulin-like growth factor binding protein-1 (IGFBP-1). Litter size was significantly reduced in Tg mice compared with wild-type mice. Immunohistochemical studies localized the expression of the transgene to the luminal and glandular epithelium. In addition, rat IGFBP-1 immunoreactivity was present in luminal secretions. E2-induced uterine DNA synthesis as measured by methyl-3H thymidine incorporation, was significantly reduced in ovex Tg mice; 4.77 +/- 0.59 and 4.97 +/- 0.53 for Tg strains 57C and 277A, respectively, compared with 8.65 +/- 0.73 fmol/microgram of DNA for Wt mice. Similarly, uterine weight after three daily injections of E2 was reduced in Tg mice compared with Wt mice; 2.85 +/- 0.39 vs. 4.23 +/- 0.26 mg/g BW, P < 0.01. Semiquantitative RT-PCR assays were used to demonstrate changes in uterine IGF-I messenger RNA (mRNA) and EGF mRNA abundance after administration of E2. An approximately 3-fold increase in IGF-I mRNA abundance was seen 6 h after E2 in both Tg and Wt mice. Over the same time course, little change was seen in EGF mRNA levels, which were similar in Tg and Wt mice. After 3 days of E2 treatment, an increase in EGF mRNA was apparent in Wt mice but not in Tg mice. The uterine DNA response to both IGF-I and EGF was significantly attenuated in Tg mice compared with Wt mice. The data reported here together with previous reports of E2 regulation of IGF-I and IGFBP-1 expression in uterus support the hypothesis that the IGF-I is a mediator of estrogen action in the uterus. In addition attenuation of the EGF response in the uterine tissue of Tg mice suggests that this response is also mediated, in part, by IGF-I.

Dexamethasone stimulation of rat insulin-like growth factor binding protein-1 (IGFBP-1) promoter activity involves the interaction of multiple transcription factors

Using an improved procedure for transient transfection of H4-II-E rat hepatoma cells, we characterized the cis elements in the proximal promoter of the rat insulin-like growth factor binding protein-1 (rat IGFBP-1) gene that are required for basal (unstimulated) and dexamethasone-stimulated promoter activity. Three sites are required for optimal basal promoter activity: an AP-2 site (nt −286 to −293), the M4 region of the insulin response element (nt −108 to −99), and a hepatocyte nuclear factor-1 (HNF-1) site (nt −62 to −50). In addition to the glucocorticoid response element (nt −91 to −77), participation of two of three accessory sites is required for optimal stimulation by dexamethasone: the M4 and HNF-1 sites, and a third site located between nt −252 and −236. Further study will focus on how the interactions of tissue-specific and hormonally-responsive transcription factors are integrated.

A novel DNA/protein complex interacts with the insulin-like growth factor binding protein-1 (IGFBP-1) insulin response sequence and is required for maximal effects of insulin and glucocorticoids on promoter function

Glucocorticoids stimulate and insulin inhibits hepatic production of IGFBP-1 at the level of gene transcription. We previously identified contiguous insulin and glucocorticoid response sequences in the proximal rat IGFBP-1 promoter. This insulin response sequence (IRS) is palindromic ( CAAAACAAA TTATTTTG) and each half resembles an IRS in the phosphoenolpyruvate carboxykinase (PEPCK) gene. We have reported that both the IGFBP-1 and PEPCK IRSs bind hepatocyte nuclear factor-3 (HNF-3) proteins [1]. We now report that IRSs from the IGFBP-1 and PEPCK, as well as an IRS which also binds HNF-3 in the rat tyrosine aminotransferase (TAT) gene, also interact with another DNA/protein complex in gel shift studies. Further, methylation interferences studies, gel shift and transient transfection studies with site-specific mutations identified a single base in the first half of the IRS that is critical both for interactions with proteins in this complex, and for maximal effects of insulin and glucocorticoids, on promoter function. Of note, a 250-fold excess of an oligo containing a C/EBP binding site (but not other AT-rich sequences) inhibits the formation of this complex in gel shift assays. Nevertheless, interactions with this C/EBP site are negligible at lower titers (≤ 100-fold excess), and antibodies against known C/EBP proteins do not react with this complex. Similarly, preincubation with CHOP, a truncated member of the C/EBP family which contains a β-leucine zipper domain, does not prevent or alter the mobility of this novel DNA/protein complex, indicating that components of this complex do not form heterodimers with β-ZIP proteins. We conclude that HNF-3 proteins and this novel C/EBP-related DNA/protein complex may play an important role in mediating interactions between glucocorticoids and insulin in the regulation of IGFBP-1 and perhaps multiple hepatic genes.

Identification of cis-elements mediating the stimulation of rat insulin-like growth factor-binding protein-1 promoter activity by dexamethasone, cyclic adenosine 3',5'-monophosphate, and phorbol esters, and inhibition by insulin.

Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates the action of IGFs on target cells. IGFBP-1 transcription is highly regulated by hormonal and metabolic factors. In rat H4-II-E hepatoma cells, IGFBP-1 messenger RNA is stimulated by dexamethasone, cAMP, and phorbol esters, and dominantly inhibited by insulin. To identify the cis-elements that determine transcriptional regulation by these agents, we have coupled rat IGFBP-1 promoter fragments to a luciferase reporter gene and transfected H4-II-E cells using the cationic liposome procedure. Promoter fragments whose 5'-end was at nucleotide (nt) -925 or -327 (with respect to the transcription initiation site, 1) conferred positive regulation of promoter activity by dexamethasone, cAMP, and phorbol esters. Insulin inhibited promoter activity in the presence of any of the three stimulatory agents. Stimulation by cAMP or phorbol esters was abolished when the region between nt -327 and -235 was deleted. Although this region contains potential activating protein-2 and activating protein-1 sites, the sites responsible for this regulation have not yet been identified. By contrast, stimulation by dexamethasone was retained in deletion constructs whose 5'-end was at nt -92, but was abolished by site mutagenesis of either the left or right half-sites of a potential glucocorticoid response element (GRE) located between nt -91 and -77. Surprisingly, substitution mutations in an up-stream region, -108 to -99 (M4), also decreased dexamethasone-stimulated promoter activity despite the presence of an intact GRE. We postulate that a positive factor that binds to the wild-type M4 region neutralizes factors that inhibit interaction of the glucocorticoid receptor with the GRE. The M4 region also is involved in inhibition by insulin. Insulin inhibition of dexamethasone-stimulated promoter activity was lost after deletion of nt -135 to -92 or mutation of the region between nt -108 and -99. This insulin response element is conserved in the human IGFBP-1 promoter and is homologous to the insulin response element of the phosphoenolpyruvate carboxykinase gene, which also is rapidly inhibited by insulin in H4-II-E cells. The rat IGFBP-1 promoter provides a valuable model system for studying the multihormonal regulation of transcription.

Dideoxy sequencing and structural analysis of the rat insulin-like growth factor binding protein-1 gene

Insulin-like growth factor binding protein-1 (IGFBP-1) is an important modulator of IGF bioavailability. To facilitate studies of IGFBP-1 regulation and function in rodent models, we cloned the rat IGFBP-1 gene and analyzed its structure by dideoxy sequencing. The rat IGFBP-1 gene is relatively small (∼ 5 kb) and contains 4 exons and 3 introns, similar to the human IGFBP-1 gene.

Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter

Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter

Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter.

Insulin-like growth factor-binding protein-1 (IGFBP-1) is produced by the liver and regulated by glucocorticoids and insulin at the level of gene transcription. To identify DNA sequences mediating the effects of glucocorticoids and insulin on IGFBP-1 promoter activity we created luciferase reporter gene constructs and performed transfection studies in H4IIE hepatoma cells. Initial studies confirmed that the IGFBP-1 promoter is functional when inserted in the correct orientation, but not in the reverse orientation. Dexamethasone (DEX) increased promoter activity 10-fold, and insulin reversed this effect of DEX by 85% at 8 h. The effects of DEX were abolished when constructs were truncated to 89 bases from the RNA cap site, and DNase footprinting with the DNA-binding domain of the human glucocorticoid receptor identified an imperfect palindrome containing two receptor-binding sites separated by three nucleotides typical of a glucocorticoid response element (GRE) at this location. Mutation of either binding site (or half-site) disrupted the effects of DEX, confirming that this sequence functions as a GRE. Two other regions of the promoter also footprinted with the glucocorticoid receptor protein and contained sequences consistent with glucocorticoid receptor-binding sites; however, neither of these footprints contained the full structure expected of a functional GRE, and neither mutation nor deletion of these other sequences altered the effects of DEX on promoter activity. To identify the DNA sequences required for the effects of insulin on glucocorticoid-stimulated promoter activity, we created internal deletions throughout the IGFBP-1 promoter region. Deletion of the 22-basepair (bp) sequence immediately 5' from the GRE disrupted the effect of insulin and appeared to increase basal promoter activity at least 2-fold in each of eight experiments (P < 0.001 vs. intact promoter). This region of the IGFBP-1 promoter contains a 19-bp palindrome (CAAAACAAACTTATTTTG) that overlaps the 5'-end of the GRE and is fully conserved in the human IGFBP-1 promoter. Each half of this palindrome resembles previously identified insulin response sequences, and deletion/mutation analysis suggests that each half may contribute to the effects of insulin on promoter activity. Gel shift studies confirmed that this palindrome binds H4IIE nuclear proteins. In summary, we have identified a GRE in the 5'-promoter region of the rat IGFBP-1 gene approximately 90 bp up-stream from the RNA cap site as well as a contiguous 22-bp region that plays a critical role in mediating the effects of insulin on glucocorticoid-stimulated promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of an insulin-responsive element in the promoter of the human gene for insulin-like growth factor binding protein-1

Insulin inhibits the hepatic transcription of insulin-like growth factor binding protein-1 (IGFBP-1). In the present studies, human HEP G2 hepatoma cells were transiently transfected with human IGFBP-1 gene promoter constructs in order to identify cis elements and trans-acting factors that confer the insulin effect. Transfections of IGFBP-1 promoter deletion constructs localized an insulin responsive element (IRE) between approximately 140- and approximately 103-base pair (bp) 5' to the mRNA capsite. This region contains a 25-bp sequence which is 100% conserved in the rat IGFBP-1 promoter and which has two AT-rich, 8-bp elements exhibiting dyad symmetry. Site-directed mutagenesis of both elements in the same 1205-bp IGFBP-1 promoter construct abolished the inhibitory effect of insulin on promoter activity. Also, the native but not the mutant IGFBP-1 IRE conferred the inhibitory effect of insulin to the heterologous thymidine kinase promoter. Gel mobility shift assays identified a DNA binding activity which specifically binds the native IGFBP-1 IRE and which is not altered by prior insulin treatment. The IGFBP-1 IRE sequence is similar to those of functionally mapped IREs from other gene promoters, suggesting that this common IRE and the protein(s) which it binds confer the insulin effect to a number of insulin-sensitive genes.

Interactive effects of nutrients and hormones on the expression of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA and peptide, and IGF I release from isolated adult rat hepatocytes.

Isolated adult rat hepatocytes were used to investigate and compare the actions of glucose or amino acids and insulin, glucagon, growth hormone, and dexamethasone on the expression of insulin-like growth factor binding protein (IGFBP) mRNA, or the release of IGFBP and IGF peptides in vitro. Ligand blot analysis of culture medium conditioned for 24 h by monolayers of hepatocytes in the presence of 6.5 mM glucose revealed two species of IGFBPs, an abundant form of 30-32 kDa and a minor species of 22-24 kDa. Western blotting showed that two IGFBPs of 29-30 and 32 kDa were recognized by antiserum against hIGFBP-1, whereas hepatocytes contained a 1.6 kb transcript on Northern blot with a rat IGFBP-1 cDNA. Insulin-like growth factor BP-2 mRNA was not detected in hepatocytes and IGFBP-2 immunoreactive peptide not present in conditioned medium. The release of IGFBP-1, determined by ligand blot, was independent of glucose concentration over the range of 2.7 mM-11.1 mM, but IGFBP-1 mRNA was decreased following incubation with 6.5 mM glucose compared with 2.7 mM glucose. The release of IGFBP-1 by hepatocytes was inhibited by insulin (10 nM-1 microM), as was mRNA abundance. However, these effects of insulin on IGFBP-1 diminished with increasing glucose concentration. Increasing concentrations of total amino acids increased IGFBP-1 release as did dexamethasone (100 pM-100 nM), whereas growth hormone and glucagon were without effect. The release of IGF I was increased by insulin, growth hormone and dexamethasone but was decreased by glucagon and amino acids, whereas changes in glucose concentration had no effect. The results show that isolated adult rat hepatocytes release IGF I and IGFBP-1 under the interactive control of nutrients and hormones involved in metabolic homeostasis.

Induction of insulin-like growth factor binding protein-1 gene expression in liver of protein-restricted rats and in rat hepatoma cells limited for a single amino acid.

Restriction of the dietary protein intake of young growing animals results in a rapid cessation of growth. In order to gain further insight into the molecular mechanisms for metabolic adaptation to protein restriction, the expression of the insulin-like growth factor binding protein-1 (IGFBP-1) gene was examined in 4-week-old male rats fed isocaloric diets containing 20%, 8%, or 4% protein over a 10-day period. Expression of the IGFBP-1 gene was strongly induced in the protein-restricted animals. Animals on the 8% protein diet exhibited a 14-fold increase, and animals on the 4% protein diet exhibited a 33-fold increase in hepatic IGFBP-1 messenger RNA (mRNA) abundance relative to the abundance of IGFBP-1 mRNA in animals on the 20% protein diet. Expression of the IGFBP-1 gene was also strongly increased by severe energy restriction: IGFBP-1 mRNA abundance was increased 15-fold in animals maintained for 10 days on a diet with energy restricted to 50% of the ad libitum intake rate. In animals fasted for 24 h there was a 6-fold increase in IGFBP-1 mRNA abundance, a lower induction than was observed in either of the two chronic nutritional restriction models. To determine whether limitation for substrate (i.e. amino acids) might have a direct effect on IGFBP-1 gene expression, we examined the effect on IGFBP-1 gene expression of limitation of H4-II-E rat hepatoma cells for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan) for a period of 24 h. The abundance of IGFBP-1 mRNA was increased by approximately 4- to 5-fold in cultures limited for any of these four amino acids as compared with its abundance in cells incubated in medium containing all essential amino acids. To study further the molecular mechanism for induction of IGFBP-1 gene expression by nutritional restriction, probes specific for intron 3 or intron 1 of the rat IGFBP-1 gene were used to quantify levels of the IGFBP-1 primary nuclear transcript in protein-restricted rats and amino acid-limited cultured cells. The level of the IGFBP-1 primary transcript was increased by 8-fold in animals on the 8% protein diet and 14-fold in animals on the 4% protein diet, suggesting that the induction of IGFBP-1 mRNA was caused largely by an increase in transcription.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of rat insulin-like growth factor-binding protein-1: the effect of insulin-induced hypoglycemia.

Rat insulin-like growth factor-binding protein-1 (rIGFBP-1) was purified from H4IIE rat hepatoma cells by IGF-I affinity chromatography and reverse-phase HPLC. A rabbit antiserum (B2) was raised to rIGFBP-1 and a RIA established. Immunoreactive IGFBP-1 was present in rat amniotic fluid and in the medium conditioned by isolated rat hepatocytes and HTC rat hepatoma cells. To study the effect of hypoglycemia, fasting female Wistar rats were anesthetized and cannulated for multiple venous sampling after the administration of insulin or saline. Serum IGFBP-1 rose in adrenal intact rats from < 0.1 micrograms/ml to a maximum of 1.41 +/- 0.23 micrograms/ml approximately 120 min after insulin administration. Compared to adrenal-intact rats, adrenalectomized animals demonstrated a delayed rIGFBP-1 response to hypoglycemia and did not appear to have reached a maximum at 180 min. A slow rise in rIGFBP-1 levels throughout the sampling period was seen after saline injection in both adrenal-intact and adrenalectomized animals. Glucose, corticosterone, rat insulin, and human insulin levels were measured and none, alone, appeared responsible for the observed rIGFBP-1 responses. We conclude that 1) rIGFBP-1 is stimulated in response to hypoglycemia in a similar manner to glucose counterregulatory hormones, 2) an adrenal factor is required for an early rIGFBP-1 response to hypoglycemia, and 3) neither circulating glucose nor insulin levels, alone, are responsible for the observed patterns of response.

Regulation of rat insulin-like growth factor-binding protein-1: the effect of insulin-induced hypoglycemia

Regulation of rat insulin-like growth factor-binding protein-1: the effect of insulin-induced hypoglycemia

Cloning of the rat insulin-like growth factor binding protein-1 gene and analysis of its 5′ promoter region

To understand specific mechanisms involved in the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), an important modulator of IGF bioactivity, we cloned the rat IGFBP-1 gene and sequenced a 1.5 kb Sph1-Sph1 fragment containing 1110 bases upstream from the translation start site. computer analysis reveals the presence of ATA, CACCC, and CCAAT elements, and putative homeodomain, AP-1, insulin and glucocorticoid response elements in the 5′ promoter. Primer extension and ribonuclease protection studies reveal a single cap site in RNA from rat hepatoma cells and both control and diabetic rat liver.

Multihormonal Regulation of Insulin-Like Growth Factor-Binding Protein-1 in Rat H4IIE Hepatoma Cells: The Dominant Role of Insulin*

Circulating levels of insulin-like growth factor-binding protein-1 (IGFBP-1) and the abundance of hepatic IGFBP-1 mRNA are increased in streptozotocin-diabetic rats and are regulated in accordance with insulin and metabolic status. We recently purified rat IGFBP-1 from medium conditioned by well differentiated rat H4IIE hepatoma cells. Since this cell line provides a useful model for examining the effects of hormones on hepatocellular function, we used H4IIE cells to examine the relative role that insulin and other factors may play in the regulation of IGFBP-1 production. H4IIE cells were stabilized in serum-free medium, then treated with specific hormones. The availability of IGFBPs in conditioned medium was estimated by [125I]IGF-I binding assay, and specific BPs were assessed by Western ligand and immunoblot analyses. The abundance of IGFBP-1 mRNA was determined by Northern and slot blot analysis. Initial studies revealed that [125I]IGF-I-binding activity in conditioned medium was reduced after 24-h incubation with 100 nM insulin (52 +/- 4% of control; P less than 0.001). In contrast, binding activity was increased after only 4 h of incubation with 75 microM 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP) or 1 microM dexamethasone (P less than 0.001 vs. control for each), but these effects were prevented by insulin. Ligand and immunoblotting demonstrated that insulin decreased the production of 32K and 34K forms of IGFBP-1, while both 8-CPT-cAMP and dexamethasone increased the production of IGFBP-1; again, insulin prevented the effects of 8-CPT-cAMP and dexamethasone. Of note, 1 microM rat GH, testosterone, progesterone, or 17 beta-estradiol had no effect on either IGF-binding activity or IGFBP-1 production. Northern and slot blot analyses revealed that 100 nM insulin profoundly lowered the abundance of IGFBP-1 mRNA in H4IIE cells (4 +/- 0.6% of control at 4 h; P less than 0.001), while IGFBP-1 mRNA was increased 2-fold during incubation with 75 microM 8-CPT-cAMP (P less than 0.001) and 9-fold with 1 microM dexamethasone (P less than 0.001). Once again, the effect of insulin was dominant; insulin both prevented and reversed the effects of maximally effective concentrations of 8-CPT-cAMP and dexamethasone. To determine whether this effect of insulin reflected altered generation or stability of IGFBP-1 mRNA, H4IIE cells were incubated with 2.5 micrograms/ml actinomycin-D with or without insulin, and mRNA was quantitated by Northern blot.(ABSTRACT TRUNCATED AT 400 WORDS)