Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cytoskeletal actins are abundant proteins in mammalian nonmuscle cells. We have previously reported that physiological concentrations of insulin induced beta-actin transcription in rat H4 hepatoma cells. To define whether one or more of the three CCArGG box elements or other elements within the beta-actin gene promoter is an insulin response element, we transfected H4 cells with regions of the human beta-actin gene promoter fused to the chloramphenicol acetyltransferase gene. A 350-basepair DNA fragment was isolated that mediates both insulin and serum effects. This fragment contains at least two up-stream elements, a CCAAT box and a CCArGG box, and accounts for more than 70% of the basal activity of the beta-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum induction, suggesting a difference in their mechanisms of action. Mutation of the CCAAT box drastically reduced basal expression, with no effect on insulin induction. In contrast, a mutation of the CCArGG element reduced basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulated the activity, but not the binding, of a factor(s) that associates with the CCArGG box. These data demonstrate that in H4 cells, insulin induction of beta-actin gene expression was mediated at least in part through one of the three beta-actin CCArGG elements.
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PMID:One of three CCArGG box/serum response elements of the beta-actin gene is an insulin-responsive element. 782 46

Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5' deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cis-acting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.
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PMID:Evidence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter activities in transiently transfected adipocytes from genetically obese rats. 783 67

Evidence from in vivo studies indicates that the bile acid pool and bile acid excretion are increased in humans with diabetes mellitus and in experimental diabetic animals, and that both parameters return to normal levels after administration of insulin. To investigate the biochemical background of these changes, the effects of insulin on bile acid synthesis and cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in routing of cholesterol toward bile acids, were studied in cultured rat hepatocytes. Mass production of bile acids was dose dependently diminished, showing significant reduction (-33% to -53%) at physiological concentrations of the hormone (1.4 to 14 nmol/L) and a maximal decrease at 140 nmol/L (-65%). The decrease of bile acid synthesis correlated well with the suppression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase activity. The enzyme activity for cholesterol 7 alpha-hydroxylase, examined in more detail, was dose dependently diminished on incubation of hepatocytes with various concentrations of insulin, reaching maximal reduction at 14 nmol/L of insulin. Maximal decrease of the enzyme activity was seen after 8 hours of incubation (-70%). Insulin strongly reduced the rise in cholesterol 7 alpha-hydroxylase activity induced by incubation with dexamethasone. Sterol 27-hydroxylase activity was inhibited up to -58% after 24 hours of incubation with 140 nmol/L insulin. To study the mechanism of suppression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase activity, the effects of insulin on their respective levels of messenger RNA (mRNA) and gene transcription were assessed. The decrease in enzyme activities could be explained by a concomitant reduction in the cholesterol 7 alpha-hydroxylase (-76%) and sterol 27-hydroxylase (-62%) mRNA level. Transcriptional activity, as assessed by nuclear runoff assays, was decreased to the same extent, i.e., -60% for cholesterol 7 alpha-hydroxylase and -75% for sterol 27-hydroxylase. Transient expression experiments using a construct containing the proximal 348 basepairs of the cholesterol 7 alpha-hydroxylase promoter fused to the chloramphenicol acetyltransferase (CAT) gene (-348Rcat) showed a significant reduction of transcriptional activity (-64%) with insulin, indicating that a sequence important for an insulin-induced transcriptional response is located within the first 348 basepairs, preceding the transcription start of the cholesterol 7 alpha-hydroxylase promoter.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Insulin suppresses bile acid synthesis in cultured rat hepatocytes by down-regulation of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase gene transcription. 784 24

Milk protein gene expression is regulated by the synergistic interactions of several lactogenic hormones, including insulin, PRL, and glucocorticoids. Whey acidic protein (WAP) gene expression is highly dependent on glucocorticoids, and to a lesser extent than casein gene expression, on the presence of PRL. Previous studies have demonstrated that a distal DNase I hypersensitive site in the rat WAP gene 5'-flanking region containing several binding sites for nuclear factor I is required for high level WAP gene expression in transgenic mice. In this study several specific glucocorticoid receptor (GR) binding sites were identified flanking these nuclear factor I sites using an in vitro DNase I footprinting assay with baculovirus-expressed GR. These sites were able to confer dexamethasone inducibility to a heterologous thymidine kinase-chloramphenicol acetyltransferase reporter gene construct in transient cotransfection experiments with GR in CV1 cells. Administration of dexamethasone to adrenal-ectomized mice carrying the +2020 rat WAP transgene during lactation demonstrated that glucocorticoids are required to maintain transgene expression in the mammary gland. Furthermore, glucocorticoid-induced changes in transgene expression were correlated with the appearance of DNase I hypersensitive sites. These results indicate that at least part of glucocorticoid regulation of WAP gene expression is mediated through the direct interaction of GR with glucocorticoid response elements in the distal promoter region resulting in steroid hormone-dependent alterations in chromatin structure.
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PMID:Glucocorticoid regulation of rat whey acidic protein gene expression involves hormone-induced alterations of chromatin structure in the distal promoter region. 785 50

We have established a system in which we observe a synergistic interaction between insulin and glucocorticoids. This includes chimeric genes constructed to contain synthetic glucocorticoid-responsive elements, 5' of the HSV thymidine kinase promoter and the chloramphenicol acetyltransferase reporter gene. The magnitude of induction of gene expression by glucocorticoid was dependent on the number of GREs. Insulin alone had virtually no effect on the expression of any of these genes but together with dexamethasone acted in a synergistic manner. This synergy diminished as the number of GREs in the promoter increased. The synergy is independent of promoter sequences other than the GREs and a functional TATAA box. Three different approaches demonstrate that the effect of insulin is not directly on the glucocorticoid signal transduction pathway. Insulin does not change the dose-response relationship for dexamethasone. The effect of insulin is independent of the intracellular concentration of glucocorticoid receptor. The effect is independent of any specific domain of the glucocorticoid receptor. The target of insulin action is likely to be part of the normal host cell transcriptional initiation complex or a putative adaptor molecule.
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PMID:Insulin enhances glucocorticoid receptor-mediated induction of gene expression independent of a specific insulin response element. 786 47

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.
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PMID:Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line. 792 31

We identified two enhancer elements of the mouse GLUT1 gene responsive to serum, growth factor, and oncogenes; the first enhancer element (enhancer-1) is located 2.7 kilobases upstream of the cap site of the gene, and the second one (enhancer-2) is located in the second intron of the gene (Murakami, T., Nishiyama, T., Shirotani, T., Shinohara, Y., Kan, M., Ishii, K., Kanai, F., Nakazuru, S., and Ebina, Y. (1992) J. Biol. Chem. 267, 9300-9306). In the present work, we describe the role of insulin in activation of these two enhancers. NIH/3T3 HIR3.5 cells, which express a large number of insulin receptors, were stably transformed by hybrid genes containing the enhancer(s) and promoter of GLUT1 gene and the coding region of chloramphenicol acetyltransferase (CAT) gene as a reporter gene. In stable transformants of the reporter gene without the enhancers, the CAT mRNA was not induced by insulin; however, in clones containing the reporter gene with enhancer-1, the CAT mRNA was induced by insulin at 30 min and reached a maximum at 1 h. In clones transfected by the reporter gene with enhancer-2, the CAT mRNA was induced at 1 h and reached a maximum at 3 h. To determine the early response element to insulin in enhancer-1, transformants of hybrid reporter genes containing truncated or mutated enhancer-1 were examined. The homologous sequence with the serum response element in enhancer-1 is essential for an early response to insulin.
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PMID:The role of insulin in activation of two enhancers in the mouse GLUT1 gene. 796 96

Many hormones regulate the rate of synthesis of phosphoenolpyruvate carboxykinase (PEPCK), the enzyme that governs the rate-limiting step in gluconeogenesis. In H4IIE rat hepatoma cells, glucocorticoids, retinoic acid and cyclic AMP (cAMP) increase PEPCK gene transcription whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant as they prevent cAMP- and glucocorticoid-stimulated PEPCK gene transcription. In contrast, insulin and phorbol esters both stimulate transcription of gene 33 in the same H4IIE cells, with the same time course as seen for their inhibitory effect on PEPCK gene transcription. We now report that the protein phosphatase inhibitor, okadaic acid, mimics the action of insulin and phorbol esters on expression of both gene 33 and PEPCK gene in H4IIE cells. Okadaic acid stimulates gene 33 mRNA accumulation whereas it inhibits cAMP- and glucocorticoid-stimulated PEPCK mRNA accumulation. The effect of okadaic acid on the PEPCK gene is mediated through the PEPCK promoter as, in a cell line, HL1C, stably transfected with a PEPCK-chloramphenicol acetyltransferase (CAT) fusion gene, okadaic acid inhibits cAMP- and glucocorticoid-stimulated CAT expression. Desensitization of the protein kinase C pathway by exposure to phorbol 12-myristate 13-acetate for 16 h abolishes the subsequent action of the phorbol ester but does not markedly affect the inhibition of cAMP- and glucocorticoid-stimulated CAT expression by insulin or okadaic acid. Even though insulin and okadaic acid appear to repress PEPCK gene expression through a pathway initially distinct from that used by phorbol esters, transient-transfection studies show that the final target of the action of okadaic acid, insulin and phorbol ester is the same DNA element.
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PMID:Comparison of the effects of insulin and okadaic acid on phosphoenolpyruvate carboxykinase gene expression. 798 Apr 40

By transfecting TSH receptor (TSHR)-chloramphenicol acetyltransferase (CAT) chimeras into FRTL-5 thyroid cells in the presence or absence of insulin, we identify an insulin-responsive element (IRE) between -220 and -190 bp of the TSHR 5'-flanking region. The region between -220 and -192 bp is footprinted by nuclear extracts from FRTL-5 cells and, coupled to a heterologous SV40-CAT chimera, an oligonucleotide containing the protected region induces insulin responsiveness in FRTL-5 cells. FRTL-5 cell nuclear extracts form two groups of protein-DNA complexes, A and B, in gel shift assays using an oligonucleotide having the protected sequence; mutation data indicate only the A complexes are increased by exposure of FRTL-5 cells to insulin; TSH can also increase A complex formation, but the TSH action is insulin-dependent. The nuclear factor(s) in FRTL-5 cells that interact with the TSHR IRE are distinct from thyroid transcription factor-2 (TTF-2), the insulin regulatory factor of the thyroglobulin promoter, as evidenced by the absence of competition in gel shift assays; there is no apparent sequence similarity of this region with other known IREs. The IRE is immediately upstream of a thyroid transcription factor-1 (TTF-1) binding site, -189 to -175 bp; mutation of the TTF-1 site causing a loss of TTF-1 activity also causes a loss of insulin responsiveness when the TSHR-CAT chimera at -220 bp is transfected into FRTL-5 cells and an altered IRE footprint by nuclear extracts. The TSHR appears, therefore, to contain a novel IRE whose activity depends at least in part on TTF-1, a thyroid-specific, homeodomain-containing transcription factor important both for thyroid-specific TSHR gene expression and TSH/cAMP autoregulation of the TSHR.
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PMID:Identification of a novel insulin-responsive element in the rat thyrotropin receptor promoter. 798 66

Glucocorticoids have been shown to increase the levels of cell surface insulin receptors and their mRNA in many different cell types. Previously, we have reported that glucocorticoids induce the transcription of the human insulin receptor (hIR) gene in rat 208F cells and we also identified a putative glucocorticoid response element (GRE) to which the glucocorticoid receptor binds in a specific manner. In this study we have mapped four additional regions of the hIR promoter to which glucocorticoid receptor binds specifically; one residues at -1340 and the others are distributed within a 100 base pair region from -750 to -650. Within each DNase I footprinting region resides at least one putative GRE sequence. They function as GREs to confer glucocorticoid inducibility when fused to a heterologous promoter-chloramphenicol acetyltransferase reporter construct. The functional significance of these putative GREs was further substantiated by mutational analysis. Taken together, our results indicate that these GREs are capable of conferring glucocorticoid-dependent transcriptional induction similar to those observed in vivo. Therefore, the increase of hIR mRNA and insulin binding to surface receptor in response to glucocorticoids is likely mediated by enhancement of transcription. The functional redundancy of the GREs may reflect the biological mechanism which ensures the glucocorticoid regulation of the hIR gene at the transcriptional level.
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PMID:Multiple hormone response elements can confer glucocorticoid regulation on the human insulin receptor gene. 805 70


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