Biomedical subjects

D S Straus

Publications and source records attributed to D S Straus.

At least 19 recordsLinked to original sources

PubMed2000

15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway.

Prostaglandin J(2) (PGJ(2)) and its metabolites Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are naturally occurring derivatives of prostaglandin D(2) that have been suggested to exert antiinflammatory effects in vivo. 15d-PGJ(2) is a high-affinity ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma) and has been demonstrated to inhibit the induction of inflammatory response genes, including inducible NO synthase and tumor necrosis factor alpha, in a PPARgamma-dependent manner. We report here that 15d-PGJ(2) potently inhibits NF-kappaB-dependent transcription by two additional PPARgamma-independent mechanisms. Several lines of evidence suggest that 15d-PGJ(2) directly inhibits NF-kappaB-dependent gene expression through covalent modifications of critical cysteine residues in IkappaB kinase and the DNA-binding domains of NF-kappaB subunits. These mechanisms act in combination to inhibit transactivation of the NF-kappaB target gene cyclooxygenase 2. Direct inhibition of NF-kappaB signaling by 15d-PGJ(2) may contribute to negative regulation of prostaglandin biosynthesis and inflammation, suggesting additional approaches to the development of antiinflammatory drugs.

Animals↗

PubMed1999

Functional activity of hepatocyte nuclear factor-1 is specifically decreased in amino acid-limited hepatoma cells.

Limitation of cultured rat hepatoma cells for an essential amino acid results in a specific decrease in expression of several genes that are preferentially expressed in the liver, including the serum albumin and transthyretin genes. In the work presented here, we examined whether the coordinate repression of these genes is caused by decreased activity of one or more of the liver-enriched transcription factors, hepatocyte nuclear factor-1 (HNF-1), HNF-3, HNF-4 or C/EBP. To address this question, HepG2 human hepatoma cells were transiently transfected with luciferase reporter constructs containing multiple copies of individual transcription factor binding sites. Limitation for an essential amino acid resulted in specific repression of a construct in which luciferase expression was directed by HNF-1. A single HNF-1 binding site located adjacent to the TATA box plays a major role in transcription directed by the serum albumin promoter in transient transfection assays. Amino acid limitation of cells transfected with an albumin promoter/luciferase reporter construct resulted in specific repression of promoter activity. In addition, bacterial methylation or site-directed mutagenesis of the HNF-1 binding site in the albumin proximal promoter region eliminated the regulation of an albumin promoter-luciferase reporter construct under conditions of amino acid limitation. These results demonstrated that the HNF-1 binding site played a major role in regulation of the albumin promoter by amino acid availability. Deletion analysis of the albumin promoter confirmed regulation through the HNF-1 binding site and also identified a second amino acid regulatory element in the upstream region of the albumin promoter, which has been shown previously to contain a functional binding site for HNF-3. The repression of albumin promoter and HNF-1 reporter constructs in amino acid-limited cells occurred without a change in the DNA binding activity of HNF-1. Moreover, HNF-3 DNA binding activity was also not decreased in amino acid-limited cells. These results suggest that the regulation of transcription by amino acids occurs at the level of transcriptional activation by HNF-1 and HNF-3, rather than by alteration of the DNA binding activity of either factor.

Amino Acids↗

PubMed1999

Upstream region of rat serum albumin gene promoter contributes to promoter activity: presence of functional binding site for hepatocyte nuclear factor-3.

Transcription of the serum albumin gene occurs almost exclusively in the liver and is controlled in part by a strong liver-specific promoter. The upstream region of the serum albumin gene promoter is highly conserved among species and is footprinted in vitro by a number of nuclear proteins. However, the role of the upstream promoter region in regulating transcription and the identity of the transcription factors that bind to this region have not been established. In the present study, deletion analysis of the rat serum albumin promoter in transiently transfected HepG2 cells demonstrated that elimination of the region between -207 and -153 bp caused a two-fold decrease in promoter activity (P<0.05). Additional analysis of the -207 to -124 bp promoter interval led to the identification of two potential binding sites for hepatocyte nuclear factor-3 (HNF-3) located at -168 to -157 bp (site X) and -145 to -134 bp (site Y). Electrophoretic mobility-shift assays performed with the HNF-3 X and Y sites demonstrated that both sites are capable of binding HNF-3alpha and HNF-3beta. Placement of a single copy of the HNF-3 X site upstream from a minimal promoter increased promoter activity by about four-fold in HepG2 cells, and the reporter construct containing this site could be transactivated if co-transfected with an HNF-3 expression construct. Furthermore, inactivation of the HNF-3 X site by site-directed mutagenesis within the context of the -261 bp albumin promoter construct resulted in a 40% decrease in transcription (P<0.05). These results indicate that the positive effect of the -207 to -153 bp promoter interval is attributable to the presence of the HNF-3 X site within this interval. Additional results obtained with transfected HepG2 cells suggest that the HNF-3 Y site plays a lesser role in activation of transcription than the X site.

Amino Acid Sequence↗

PubMed1999

Ifosfamide, carboplatin, and etoposide: a highly effective cytoreduction and peripheral-blood progenitor-cell mobilization regimen for transplant-eligible patients with non-Hodgkin's lymphoma.

PURPOSE: To evaluate a chemotherapy regimen that consisted of ifosfamide administered as an infusion with bolus carboplatin, and etoposide (ICE) supported by granulocyte colony-stimulating factor (G-CSF) for cytoreduction and stem-cell mobilization in transplant-eligible patients with primary refractory or relapsed non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: One hundred sixty-three transplant-eligible patients with relapsed or primary refractory NHL were treated from October 1993 to December 1997 with ICE chemotherapy at Memorial Sloan-Kettering Cancer Center. Administration of three cycles of ICE chemotherapy was planned at 2-week intervals. Peripheral-blood progenitor cells were collected after cycle 3, and all patients who achieved a partial response (PR) or complete response (CR) to ICE chemotherapy were eligible to proceed to transplantation. Event-free and overall survival, ICE-related toxicity, and the number of CD34(+) cells collected after treatment with ICE and G-CSF were evaluated. RESULTS: All 163 patients were assessable for response, and there was no treatment-related mortality. A major response (CR/PR) was evident in 108 patients (66.3%); 89% of the responding patients underwent successful transplantation. Patient who underwent transplantation and achieved a CR to ICE had a superior overall survival to that of patients who achieved a PR (65% v 30%; P =.003). The median number of CD34(+) cells/kg collected was 8.4 x 10(6). The dose-limiting toxicity of ICE was hematologic, with 29.4% of patients developing grade 3/4 thrombocytopenia. There were minimal nonhematologic side effects. CONCLUSION: ICE chemotherapy, with ifosfamide administered as a 24-hour infusion to decrease CNS side effects, and the substitution of carboplatin for cisplatin to minimize nephrotoxicity, is a very effective cytoreduction and mobilization regimen in patients with NHL. Furthermore, the quality of the clinical response to ICE predicts for posttransplant outcome.

Adolescent↗

PubMed1998

Effects of cyclopentenone prostaglandins and related compounds on insulin-like growth factor-I and Waf1 gene expression.

The molecular pathways by which the cyclopentenone prostaglandins (PGA and PGJ series) inhibit cell growth and tumorigenicity are poorly understood. These cellular responses may be caused by specific regulation of growth-related and stress-induced genes. A variety of prostaglandins were tested for their ability to regulate insulin-like growth factor-I (IGF-I) and Waf1 gene expression in C6 rat glioma cells. The prostaglandins (in order of potency) PGJ2 > PGA1 > PGA2, approximately PGD2 >> PGE2 all significantly repressed IGF-I gene expression. With the exception of PGE2, the same prostaglandins that repressed IGF-I also induced Waf1 gene expression. However, the order of potency for Waf1 induction was different than for IGF-I repression: PGA2 > PGA1 approximately PGJ2 > PGD2. The different order of potency of the prostaglandins in regulating IGF-I and Waf1 gene expression suggests that different intracellular signals may be involved in regulating the two genes. Augmentation of glutathione levels by pretreatment of cells with N-acetyl-L-cysteine attenuated the effect of PGA2 on IGF-I and Waf1 gene expression. conversely, depletion of the intracellular glutathione pool by pretreatment with buthionine sulfoximine potentiated the effect of PGA2 on the expression of both genes. These results suggest that conjugation with glutathione prevents the regulation of gene expression by PGA2. We also tested the effect of several simpler compounds that contain a five-membered ring system on IGF-I and Waf1 gene expression. 2-Cyclopenten-1-one, but not cyclopentene or cyclopentene, repressed IGF-I and induced Waf1 gene expression, demonstrating the requirement for an alpha, beta-unsaturated carbonyl for regulation of the two genes. The dione compound 4-cyclopentene-1,3-dione, which has two potentially reactive carbons rather than one, was considerably more potent than 2-cyclopentene-1-one in repressing IGF-I gene expression (IC50 = 30 microM for 4-cyclopentene-1,3-dione as compared with 167 microM for 2-cyclopentene-1-one). Additional results indicated that diethyl maleate, which has two alpha,beta-unsaturated carbonyls in a non-cyclic configuration, also repressed IGF-I gene expression (IC50 = 214 microM) and induced Waf1 gene expression, indicating that the cyclic structure is not required for either effect.

Animals↗

PubMed1998

LDL induces transcription factor activator protein-1 in human endothelial cells.

Low density lipoprotein (LDL) has been shown to perturb endothelial cells, with manifestations ranging from alterations in free radicals and arachidonate metabolism to stress fiber formation and monocyte recruitment. Some of these changes are regulated by LDL at the transcriptional level. Using mobility shift assays with consensus sequences for various transcription factors, we have detected an increase in activator protein 1 (AP-1), but not nuclear factor-kappaB (NF-kappaB), binding in human umbilical vein endothelial cells exposed to LDL. Following transfection, AP-1-driven chloramphenicol acetyltransferase and AP-1-driven-luciferase are upregulated by LDL. In contrast, there is no effect on NF-kappaB-driven chloramphenicol acetyltransferase. AP-1 increases in a biphasic fashion, with the first peak occurring 6 hours after and the second 48 hours after exposure to LDL. This AP-1 binding increase involves c-Jun, but not c-Fos, as shown by gel supershift, Northern hybridization, and Western blotting analyses. c-Jun mRNA levels are elevated by 9 hours after and remain so until at least 24 hours after exposure to LDL. c-Jun protein levels increase at 12 hours and continue to rise for 24 hours after exposure to LDL. Moreover, this LDL-increased AP-1 binding is suppressed by several protein kinase (PK) inhibitors: the PKC inhibitor calphostin C, the cAMP-dependent PK inhibitor H89, and the tyrosine PK inhibitors genistein and lavendustin A. This study demonstrates that (1) LDL is an endothelial agonist distinct from other cell stimulators, such as cytokines, endotoxin, and phorbol 12-myristate 13-acetate, because LDL appears to activate human umbilical vein endothelial cells predominantly through the transcription factor AP-1 and not NF-kappaB; and (2) LDL increases AP-1 via mechanisms involving multiple kinase activities and c-Jun transcription.

Animals↗

PubMed1997

Prostaglandin A2 specifically represses insulin-like growth factor-I gene expression in C6 rat glioma cells.

The cyclopentenone PGs (PGA and PGJ series) inhibit tumor cell proliferation in vitro and tumorigenesis in vivo via mechanisms that are at present poorly understood. The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which is believed to act as an autocrine factor for these cells. PGA2 inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G1 phase of the cell cycle. The inhibition of cell proliferation by PGA2 is accompanied by a decrease in the abundance of IGF-I messenger RNA (mRNA). This regulation of IGF-I gene expression is specific, as the abundance of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and ubiquitin mRNA is not significantly affected by PGA2. The repression of IGF-I gene expression is observed at PGA2 concentrations as low as 10 microM and is evident within 4 h after treatment of the C6 cells with PGA2. In addition to specifically regulating the expression of the IGF-I gene, PGA2 also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA2 is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA2 action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA2, we developed a sensitive RT-PCR assay for IGF-I nuclear transcripts. A similar assay was developed for quantifying HPRT transcripts, which were used as a control. Treatment of the C6 cells with 20 microM PGA2 resulted in approximately a 6-fold decrease in IGF-I mRNA and IGF-I nuclear transcripts. In contrast, HPRT mRNA and nuclear transcript levels were not significantly affected by PGA2. These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA2 is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA2 on IGF-I transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase transcription is driven by IGF-I P1 promoter fragments extending from -1711 to -328 or from -1114 to +328 relative to the beginning of exon 1. Treatment of cells with PGA2 in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA2 is located outside the -1711 to +328 promoter interval.

Animals↗

PubMed1996

Effect of dietary protein restriction on liver transcription factors.

The transcription of several genes that are preferentially expressed in the liver, including the serum albumin, transthyretin and carbamyl phosphate synthetase-I genes, is specifically decreased in animals consuming inadequate amounts of dietary protein. The high level of transcription of these genes in the liver is directed in part by a number of liver-enriched transcription factors, including hepatocyte nuclear factors (HNF)-1, -3, and -4, and proteins of the CCAAT/enhancer-binding protein (C/EBP) family. In the present study, we investigated the possibility that the co-ordinate decrease in transcription of the nutritionally sensitive genes in protein-deprived rats results from altered activity of one or more of the liver-enriched transcription factors. For HNF-4, Western blots indicated no change in the level of nuclear HNF-4 protein in liver of protein-deprived animals, whereas we observed a 40% reduction in the DNA binding activity of HNF-4 as measured by electrophoretic mobility shift assay (EMSA). Furthermore, the binding affinity of HNF-4 for DNA was unaltered by dietary protein deprivation, while the number of HNF-4 molecules able to bind to DNA (Bmax) was reduced, as determined by Scatchard analysis. This indicates that in the protein-restricted rats a portion of the pool of HNF-4 protein is inactivated or otherwise prevented from binding to DNA. The overall DNA binding activity of C/EBP alpha and beta was increased in protein-restricted animals. This change occurred in the absence of a change in the amount of the full-length forms of these two proteins, quantified by Western blotting. Interestingly, dietary protein restriction specifically increased the level of a truncated form of C/EBP beta (liver-enriched transcriptional inhibitory protein, LIP), which is a protein dominant negative inhibitor of C/EBP function. Analysis of HNF-3 DNA-binding activity by EMSA revealed that HNF-3 alpha and beta DNA binding was increased and that HNF-3 gamma DNA-binding activity was unchanged in protein-restricted animals. We also detected two apparently novel shift complexes with the HNF-3 probe by EMSA, both of which were decreased in protein-restricted animals. HNF-1 DNA-binding activity was increased by dietary protein restriction. We also examined the effect of protein restriction on the DNA-binding activity of two ubiquitous transcription factors, NF1 and Sp1. The DNA binding activity of the major NF1 isoforms was unchanged whereas the binding activity of Sp1 was increased in the protein-restricted animals. In summary, restriction of dietary protein resulted in a number of specific changes in the DNA-binding activity of various transcription factors. Because transcriptional activation typically involves the synergistic action of more than one transcription factor, small changes in the amount/activity of several factors, could have a strong net effect on the transcription of many genes.

Animals↗

PubMed1995

Glucose stimulates IGF-I gene expression in C6 glioma cells.

Glucose stimulates expression of the insulin-like growth factor I (IGF-I) gene in cultured C6 glioma cells. This stimulation is specific, as the expression of other genes, including those encoding hypoxanthine guanine phosphoribosyl transferase (HPRT) and ubiquitin, is not similarly affected by glucose. IGF-I gene expression is also stimulated by lactate, suggesting that the stimulatory effect is mediated by a product of glycolysis. Additional results indicate that the abundance of IGF-I mRNA is considerably higher in stationary confluent cells than in log-phase growing cells. This regulation is also specific for IGF-I, as HPRT mRNA is regulated in the opposite direction.

Animals↗

PubMed1995

IGF-I and serine protease inhibitor 2.1 nuclear transcript abundance in rat liver during protein restriction.

Restriction of dietary protein consumption of young male rats results in decreased growth velocity and a reduction in the abundance of hepatic IGF-I mRNA. It is not known whether the reduction of IGF-I mRNA abundance in the liver of protein-restricted rats results from a decrease in IGF-I gene transcription. In the present study, three experiments were performed with 4-week-old male rats to examine the effect of protein restriction on IGF-I gene transcription in liver. In these experiments, we monitored IGF-I nuclear transcripts (pre-mRNA) within total cellular RNA using a ribonuclease protection assay. In the first experiment, a consistent decrease in IGF-I mRNA from animals fed isocaloric diets containing 20% (control), 12%, 8% and 4% protein (dietary effect, P < 0.001) was not paralleled by a decrease (P > 0.50) in IGF-I pre-mRNA. Two additional experiments examining the effect of 4% vs 20% protein diets yielded comparable results. Pooled results from these two studies (n = 12/treatment) demonstrated that a 64% reduction (P < 0.0001) in IGF-I mRNA abundance was not accompanied by a decrease in IGF-I pre-mRNA (1.17 vs 1.31 +/- 0.21 image density units for 4% and 20% protein treatments). Unlike IGF-I, the abundance of carbamyl phosphate synthetase-I (CPS-I) pre-mRNA and mRNA was comparably reduced (approximately 70%, P < 0.001), indicating that the decrease in mRNA of this urea cycle enzyme during protein restriction occurs predominantly by a transcriptional mechanism. A common feature of all experiments was a pronounced variability in the expression of hepatic IGF-I pre-mRNA among animals, which was not diet specific. To test whether the variability in IGF-I gene transcription was correlated with variability in the transcription of another gene that is regulated by GH, we quantified the abundance of nuclear transcripts for the serine protease inhibitor 2.1 (SPI 2.1) gene. A positive association (r = 0.81, P < 0.0001) between SPI 2.1 and IGF-I nuclear transcripts was demonstrated. The correlation between IGF-I and SPI 2.1 transcripts was specific, because the quantity of IGF-I and CPS-I nuclear transcripts was not correlated in this study. Although transcription of the IGF-I and SPI 2.1 genes was similar, the abundance of SPI 2.1 mRNA was not altered by protein deprivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Protein restriction specifically decreases the abundance of serum albumin and transthyretin nuclear transcripts in rat liver.

The expression of the genes for serum albumin and several other plasma proteins is decreased in animals consuming inadequate amounts of dietary protein. To define the specificity of this phenomenon, we examined the effect of dietary protein restriction on the abundance of the mRNA for nine genes in rat liver. The results of this and previous studies indicate that genes in liver can be divided into two classes based on their response to protein restriction. Group I genes (albumin, transthyretin, carbamyl phosphate synthetase-I, class I alcohol dehydrogenase, insulin-like growth factor-I) exhibit decreased expression in response to protein restriction. In contrast, the expression of group II genes (hypoxanthine-guanine phosphoribosyl transferase, ubiquitin, H-ferritin, insulin-like growth factor binding proteins-1, -2 and -4) is either unchanged or increased in response to protein restriction. To investigate the molecular mechanism(s) leading to the decreased level of albumin and transthyretin mRNA in protein-restricted animals, the effect of protein restriction on the abundance of albumin and transthyretin nuclear transcripts was examined. The results demonstrated that protein restriction specifically decreased the abundance of albumin and transthyretin nuclear transcripts, indicating that the reduction in mRNA levels is caused at least partly by a decrease in gene transcription.

Animals↗

PubMed1994

Nutritional regulation of hormones and growth factors that control mammalian growth.

Juvenile animals stop growing if they are fed a diet containing an inadequate amount of energy or protein. The molecular basis for this growth arrest is not completely understood. The cessation of growth that occurs in nutritionally restricted animals is not generally explained by a decrease in circulating growth hormone (GH). In most species, plasma GH is increased rather than decreased under conditions of nutritional restriction. Current evidence suggests that the biosynthesis of insulin-like growth factor-I (IGF-I) is a key control point for nutritional regulation of growth. Plasma IGF-I peptide levels and hepatic IGF-I mRNA abundance are correlated with growth velocity and are consistently decreased when growth is arrested by nutritional deprivation. The decreased IGF-I mRNA abundance observed in the fasting rat appears to be caused primarily by a decrease in IGF-I gene transcription. In tissues and plasma, the insulin-like growth factors are complexed with high-affinity binding proteins, which are thought to modulate the tissue access and action of the IGFs. The hepatic mRNA abundance of two of the binding proteins (IGFBP-1 and -2) is increased in nutritionally restricted animals. This increase in mRNA abundance is caused primarily by an increase in transcription of the IGFBP-1 and IGFBP-2 genes. Current research is focused on molecular mechanisms for regulation of IGF-I and IGF-binding protein gene expression.

Animals↗

PubMed1994

Effect of amino acid limitation on the expression of 19 genes in rat hepatoma cells.

We showed previously that the abundance of serum albumin mRNA is decreased in H4-II-E rat hepatoma cells limited for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan). To define the specificity of this phenomenon, we examined the effect of amino acid limitation on the abundance of mRNAs for 19 genes in the H4-II-E cells. These genes included six genes whose expression is either completely liver-specific or highly enriched in the liver compared with other tissues [albumin, transthyretin (TTR), transferrin, carbamyl phosphate synthetase-I, urate oxidase, class I alcohol dehydrogenase], as well as a number of ubiquitously expressed "housekeeping" genes. The results indicated that the 19 genes could be divided into three classes based on their response to amino acid limitation. Class I genes (the six liver-specific genes and alpha-tubulin) exhibit decreased expression in response to amino acid limitation. The expression of class II genes [beta 2-microglobulin, hypoxanthine-guanine phosphoribosyl transferase (HPRT), H-ferritin, ubiquitin (UbB), insulin-like growth factor binding protein-4, HNF-1 alpha] is not significantly affected by amino acid limitation. Class III genes [gadd153, beta-actin, ubiquitin (UbC), phosphoglycerate kinase-1, C/EBP alpha, C/EBP beta] exhibit increased expression in response to amino acid limitation. Thus, specific inductive as well as repressive effects on gene expression are quite common in amino acid-limited cells. The observation that all six genes whose expression is liver-specific exhibited decreased expression in amino acid-limited cells suggests a common mode of regulation of these genes by amino acid availability. The strong induction by amino acid limitation of the C/EBP inhibitor gadd153 is of interest in this regard, as increased levels of gadd153 could interfere with C/EBP, which is required for high expression of most liver-specific genes. To investigate further the molecular mechanism for the decrease in albumin mRNA abundance, albumin nuclear transcript levels were quantified in control and tryptophan-limited cells. Tryptophan limitation caused a decrease in albumin nuclear transcript abundance, and this decrease preceded the decrease in albumin mRNA, suggesting that the decrease in albumin mRNA was caused at least partly by a decrease in albumin gene transcription. Additional experiments with actinomycin D indicated that albumin mRNA was also destabilized in the tryptophan-limited cells. Thus, the overall results indicate that the decrease in albumin mRNA in the tryptophan-limited cells is caused by a specific decrease in albumin nuclear transcript abundance and destabilization of albumin mRNA.

Amino Acids↗

PubMed1994

The effect of fasting on insulin-like growth factor-I nuclear transcript abundance in rat liver.

The abundance of insulin-like growth factor I (IGF-I) messenger RNA (mRNA) is decreased in the liver of fasting, protein-restricted, and energy-restricted rats. The extent to which this decrease in steady state mRNA abundance may be attributed to a decrease in IGF-I gene transcription remains unresolved. In the present study, we used an RNase protection assay to quantify IGF-I nuclear transcript (pre-mRNA) and mRNA abundance in whole cellular RNA isolated from liver of fasted and nonfasted male rats (4-6 weeks of age). The results of the RNase protection assay of IGF-I nuclear transcripts were strongly correlated with the results of nuclear transcription elongation (run-on) assays (r > 0.90; P < 0.001). In addition, the RNase protection assay allows for a greater capability for sensitively monitoring gene transcription in a large number of samples. In four different experiments, a consistent decrease in the quantity of IGF-I nuclear transcripts was observed in liver of animals fasted for 72 h, whereas IGF-I pre-mRNA abundance in animals fed ad libitum was highly variable (average intraassay coefficient of variation = 74% vs. 34% for nonfasted and fasted groups). When data from the four experiments were pooled, fasting reduced IGF-I pre-mRNA and mRNA levels by 78% and 70% (P < 0.001), respectively. Fasting also caused a significant decrease in mRNA and nuclear transcript abundance for another nutritionally sensitive gene, the gene encoding transthyretin (TTR). To determine whether the decrease in IGF-I and TTR nuclear transcripts was gene specific, levels of nuclear transcripts for serum albumin, H-ferritin, and ribosomal RNA were also quantified. The results indicated that serum albumin, H-ferritin, and ribosomal RNA nuclear transcripts were not decreased by fasting, demonstrating that the negative effect of fasting was specific for IGF-I and TTR. In summary, these results indicate that IGF-I and TTR nuclear transcripts are specifically decreased by fasting. The decrease in IGF-I mRNA is matched by a similar decrease in IGF-I nuclear transcripts, suggesting that fasting controls IGF-I gene expression primarily at the transcriptional level.

Animals↗

PubMed1993

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)

Amino Acid Sequence↗

PubMed1992

Transcription of the insulin-like growth factor-binding protein-2 gene is increased in neonatal and fasted adult rat liver.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of proteins that specifically bind IGF-I and IGF-II, determine their bioavailability to tissues, and modulate their actions in target tissues. Levels of IGFBPs in plasma and IGFBP mRNAs in liver are highly regulated with developmental age and metabolic status. We now demonstrate that the increase in IGFBP-2 mRNA in fasted adult rat liver and in the liver of normal neonatal rats reflects an increased rate of transcription. When adult rats were fasted for 2-3 days, IGFBP-2 mRNA was increased in liver, but not in brain or kidney. The increase in hepatic IGFBP-2 mRNA was observed after only 1 day of fasting. Levels decreased by half after 6 h of refeeding and returned to their low starting values after 2 days of refeeding. Transcription-elongation experiments indicated that transcription of the IGFBP-2 gene was increased in fasted liver. The rate of transcription increased 9.2- +/- 3.5-fold for transcripts labeled in exon 1 and 6.6- +/- 2.4-fold for transcripts labeled in exons 2, 3, and 4, suggesting that fasting causes a uniform increase in the number of RNA polymerase II molecules along the length of the IGFBP-2 gene. We infer from these results that the regulation of IGFBP-2 gene transcription in fasting occurs at the level of initiation rather than elongation. IGFBP-2 gene transcription also was increased 3.8- +/- 1.2-fold (exon 1) and 2.9- +/- 0.9-fold (exons 2, 3, and 4) in nuclei from 2-day postnatal rat liver compared with adult rat liver, consistent with the greater abundance of IGFBP-2 mRNA in neonatal rat liver.

Animals↗

PubMed1991

Specific decrease in liver insulin-like growth factor-I and brain insulin-like growth factor-II gene expression in energy-restricted rats.

Four-week-old male rats were maintained for 10 d on a series of diets containing a constant high level of dietary protein and total energy at 100, 70, 60 or 50% of the ad libitum intake rate. Under these conditions, growth rate varied as a function of dietary energy. Serum insulin-like growth factor (IGF)-I was decreased in the energy-restricted animals. Total hepatic IGF-I mRNA was decreased by approximately the same factor as circulating IGF-I protein. In contrast to previous results obtained with protein-restricted animals, serum albumin mRNA was not decreased in the energy-restricted animals. Brain IGF-II mRNA was slightly decreased in animals fed the 70 and 60% energy diets and was decreased by 50% in animals fed the 50% energy diet. Insulin-like growth factor binding protein-2 (IGFBP-2) gene expression was increased in the liver but not in the brain of the energy-restricted animals, indicating that dietary energy regulates IGFBP-2 gene expression differently in liver and brain. The results demonstrate specific changes in liver IGF-I and IGFBP-2 gene expression and brain IGF-II gene expression in animals that are growth-retarded because of a restriction of dietary energy.

Animals↗

PubMed1991

Expression of the genes for insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding proteins-1 and -2 in fetal rat under conditions of intrauterine growth retardation caused by maternal fasting.

Evidence suggests that insulin-like growth factors-I and -II (IGF-I and II) play a role in regulating fetal growth and development. In the fetus, IGF-I and -II are complexed with two specific binding proteins (IGFBP-1 and -2), which are thought to modulate the actions of the IGFs in target tissues. We examined regulation of the genes for IGF-I, IGF-II, IGFBP-1, and IGFBP-2 in fetal rat liver in an experimental model for intrauterine growth retardation caused by maternal fasting on days 17-21 of gestation. The mean weight of fetuses from the fasted dams was 27-32% lower than the mean weight of fetuses from the fed dams. The concentration of immunoreactive IGF-I was decreased by 71% in serum of fetuses from the fasting dams. The concentration of immunoreactive IGF-II was slightly decreased (by 12%) in serum of fetuses from the fasting dams, whereas the concentration of immunoreactive pro-IGF-II E-domain peptide was decreased by 31%. The abundance of hepatic IGF-I mRNA was decreased by 55% in fetuses from the fasting dams. In contrast, the abundance of IGF-II mRNA in fetal liver was not significantly decreased by maternal fasting. Maternal fasting caused a 2-fold increase in the abundance of IGFBP-1 mRNA in fetal liver, whereas it did not change the abundance of IGFBP-2 mRNA. The induction of IGFBP-1 mRNA in liver of the growth-retarded fetuses is similar to the induction that occurs in liver of fasting adults, while the lack of regulation of IGFBP-2 mRNA differs from the strong induction of IGFBP-2 mRNA that occurs in liver of fasting adults. In summary, these results indicate that maternal fasting causes a decrease in fetal IGF-I gene expression, a decrease in fetal serum IGF-I, and a slight decrease in fetal serum IGF-II and pro-IGF-II E-domain peptide concentrations. Maternal fasting also causes an increase in fetal IGFBP-1 gene expression. Changes in fetal insulin and glucose may be related to changes in expression of the IGF-I and IGFBP-1 genes in the growth-retarded fetuses. The decreased expression of IGF-I and -II and increased expression of the IGFBP-1 gene may contribute to the fetal growth retardation observed in this model system.

Animals↗