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R W Hanson

Publications and source records attributed to R W Hanson.

At least 19 recordsLinked to original sources
PubMed1998

Nuclear factor I regulates expression of the gene for phosphoenolpyruvate carboxykinase (GTP).

Nuclear factor-I (NFI) binds to the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter immediately 5' to the cAMP regulatory element (CRE). This suggests an interaction between NFI and factors that bind the CRE. Of the four NFI isoforms expressed in mammalian tissues, NFI-A and -B stimulate basal transcription from the PEPCK gene promoter in HepG2 cells, while NFI-C and -X are slightly inhibitory. All four NFI isoforms abrogate the 20-fold protein kinase Ac (PKAc)-mediated induction of transcription from the PEPCK gene promoter. Normal PKAc-mediated induction was noted when the CRE was moved 10 base pairs 3' of its original location. However if the CRE was moved 5 base pairs 3', placing it out of phase with the other elements in the promoter, or moved 5' to -285 (the P3(I) site in the promoter), some PKA-mediated stimulation was lost. The NFI-C isoform effectively inhibited PKAc induction regardless of the relative positions of the CRE and the NFI binding sites. NFI-C also abrogated cAMP regulatory element-binding protein (CREB)-induced activity of wild type and mutant PEPCK promoters. There was some cooperativity in the binding of CREB and NFI to their respective binding sites but this did not appear to be physiologically important.

Animals↗
PubMed1998

Glycemia-lowering effect of cobalt chloride in the diabetic rat: role of decreased gluconeogenesis.

Results of previous studies indicated that treatment of diabetic rats (induced by streptozotocin) with cobalt chloride (CoCl2) resulted in a significant decrement in serum glucose concentration. The present study was designed to determine the potential role of enhanced glucose uptake vs. decreased glucose production in the above response. The rate of systemic appearance of glucose, measured under fasting conditions using [3-3H]glucose tracer, was reduced from 35.5 +/- 2.5 to 17.5 +/- 1.8 micromol . kg-1 . min-1 in diabetic rats treated with 2 mM CoCl2 added to the drinking water for 10-14 days (P < 0.01). Tissue accumulation of intravenously administered 2-deoxy-[14C]glucose was significantly reduced in kidney and eye of diabetic rats treated with CoCl2, whereas the uptake remained unchanged in several other tissues including cerebrum, red and white skeletal muscle, heart, and liver. The relative content of phosphoenolpyruvate carboxykinase (PEPCK) mRNA was increased 3.1-fold in livers of diabetic compared with normal rats (P < 0.001), and treatment of diabetic rats with CoCl2 decreased hepatic PEPCK mRNA levels to normal. The content of PEPCK mRNA in the liver was decreased by 33% in CoCl2-treated normal rats (P < 0.05). Treatment with CoCl2 resulted in no change in cAMP levels in the livers of either diabetic or normal rats. These results suggest that the glycemia-lowering effect of CoCl2 is mediated by reductions in the rate of systemic appearance of glucose and hepatic gluconeogenesis.

Animals↗
PubMed1997

Role of the isoforms of CCAAT/enhancer-binding protein in the initiation of phosphoenolpyruvate carboxykinase (GTP) gene transcription at birth.

The gene for phosphoenolpyruvate carboxykinase (PEPCK), a target of CCAAT/enhancer-binding protein-alpha (C/EBPalpha) and -beta (C/EBPbeta), begins to be expressed in the liver at birth. Mice homozygous for a deletion in the gene for CEBPalpha (C/EBPalpha-/- mice) die shortly after birth of hypoglycemia, with no detectable hepatic PEPCK mRNA and negligible hepatic glycogen stores. Half of the mice homozygous for a deletion in the gene for CEBPbeta (C/EBPbeta-/- mice) have normal glucose homeostasis (phenotype A), and the other half die at birth of hypoglycemia due to a failure to express the gene for PEPCK and to mobilize hepatic glycogen (phenotype B). Insulin deficiency induces C/EBPalpha and PEPCK gene transcription in the livers of 19-day fetal rats, whereas dibutyryl cyclic AMP (Bt2cAMP) increases the expression of the gene for C/EBPbeta and causes a transient burst of PEPCK mRNA. Bt2cAMP induces PEPCK mRNA in the livers of fetal C/EBPalpha-/- mice, but at only 20% of the level of control animals; however, there is no induction of PEPCK mRNA if the cyclic nucleotide is injected into C/EBPalpha-/- mice immediately after delivery. The expression of the gene for C/EBPbeta is markedly induced in the livers of C/EBPalpha-/- mice within 2 h after the administration of Bt2cAMP. C/EBPbeta-/- mice injected at 20 days of fetal life with Bt2cAMP have a normal pattern of induction of hepatic PEPCK mRNA. In C/EBPbeta-/- mice with phenotype B, the administration of Bt2cAMP immediately after delivery induces PEPCK mRNA, causes the mobilization of hepatic glycogen, and maintains normal glucose homeostasis for up to 4 h (duration of the experiment). We conclude that C/EBPalpha is required for the cAMP induction of PEPCK gene expression in the liver and that C/EBPbeta can compensate for the loss of C/EBPalpha if its concentration is induced to appropriate levels.

Animals↗
PubMed1997

Involvement of HNF-1 in the regulation of phosphoenolpyruvate carboxykinase gene expression in the kidney.

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene is differentially expressed in several tissues. A specific set of regulatory elements in the promoter are responsible for the control of PEPCK gene transcription and, in turn, determine its distinct metabolic role in each tissue. DNase I footprinting analysis of the PEPCK promoter, using nuclear proteins from tissues which express the gene for PEPCK, and transient expression assays in renal cell lines have demonstrated that the HNF-1 recognition motif (P2) in the PEPCK promoter characterizes kidney-specific expression. This site is required also for the response to acidosis. Since the P2 site is not involved in the expression of the PEPCK gene in the liver, we propose that its critical role in the kidney stems from a combination of abundance of HNF-1 together with low concentrations of members of the C/EBP family in this tissue.

Acids↗
PubMed1997

Biochemical and functional characterization of DNA complexes capable of targeting genes to hepatocytes via the asialoglycoprotein receptor.

Electrostatic binding of polycations or basic polypeptides to the DNA phosphate backbone has been previously described as a one-step process which results in uncontrolled aggregation and precipitation of the DNA in solution. We describe here a multistep process in which the condensation of DNA in the presence of poly-L-lysine can be controlled to produce particles of discrete size and shape suitable for receptor-mediated gene transfer in vivo and in vitro. The first step in this process involves the gradual accretion of poly-L-lysine onto the DNA phosphate backbone, until charges are neutralized. The addition of poly-L-lysine to a concentrated solution of DNA in this fashion prevents intermolecular aggregation of the DNA, presumably by promoting the formation of a nucleus of condensation along the length of each DNA molecule. The second stage of the process involves adjusting the ionic strength of the solvent to facilitate the solubilization of compact DNA.poly-L-lysine complexes. Several physical and biochemical parameters have been studied and correlated with the efficacy of DNA/ligand-poly-L-lysine particles in transferring genes to the liver of adult animals by receptor-mediated endocytosis.

Asialoglycoprotein Receptor↗
PubMed1997

Axon-mediated gene transfer of retinal ganglion cells in vivo.

Modification of the intracellular functions of mature neurons through specific gene transfer has many potential applications. Here we present a new methodology for the successful transfection of retinal ganglion cells by administration of plasmid at the cut end of the optic nerve, or at their intact axon terminals; the latter is significantly more efficient. Plasmids contained either the SV40 promoter linked to the luciferase gene, or the CMV or RSV promoter linked to the lacZ gene. Assays for both reporter genes demonstrated significant expression of exogenous DNA in the retina for at least 10 days after retrograde transport. Duration of expression was extended to 20 days or more (duration of the experiment) when plasmid DNA was condensed with poly(L-lysine). beta-Galactosidase analysis revealed transfection of ganglion cells in high numbers. Such an approach for gene delivery to specific subpopulations of neurons might be useful in studies of molecular functions in vivo and as an experimental therapeutic strategy to extend survival and restore their function.

Animals↗
PubMed1997

The promoter regulatory regions of the genes for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken and the rat have different species-specific roles in gluconeogenesis.

Hepatic expression of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) in birds occurs prior to birth and decreases to negligible levels before hatching, whereas in mammals the gene for PEPCK-C in the liver is expressed at birth and is active throughout the life of the animal. The administration of cyclic AMP to adult chickens results in the induction of transcription of the gene for PEPCK-C and the transient accumulation of PEPCK-C mRNA in the liver. DNase I footprint analysis of 330 bp of the avian PEPCK-C promoter immediately 5' of the start-site of transcription indicated the presence of several protein binding domains, purified CAAT/enhancer binding protein alpha, cAMP regulatory element binding protein and nuclear factor-1 bound to these regions of the promoter. Sequences corresponding to an hepatic nuclear factor-1 binding domain and to the insulin response sequence, previously identified in the rat PEPCK-C promoter, were also found in the chicken PEPCK-C promoter. Co-transfection of an expression vector for CAAT/enhancer binding protein alpha or CAAT/enhancer binding protein beta markedly stimulated transcription from both the chicken and rat PEPCK-C promoters in human hepatoma cells. Sequences involved in the regulation of gene transcription by cyclic AMP and insulin were found to reside between -210 and +1 of the avian PEPCK-C promoter. In general, transcription from the avian promoter was more sensitive to inhibition by insulin than was noted for the rat PEPCK-C promoter, which may explain in part the lack of expression of the gene for PEPCK-C in the livers of adult birds.

Animals↗
PubMed1997

Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression.

Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is a key enzyme in the synthesis of glucose in the liver and kidney and of glyceride-glycerol in white adipose tissue and the small intestine. The gene for the cytosolic form of PEPCK (PEPCK-C) is acutely regulated by a variety of dietary and hormonal signals, which result in alteration of synthesis of the enzyme. Major factors that increase PEPCK-C gene expression include cyclic AMP, glucocorticoids, and thyroid hormone, whereas insulin inhibits this process. PEPCK-C is absent in fetal liver but appears at birth, concomitant with the capacity for gluconeogenesis. Regulatory elements that control transcription of the PEPCK-C gene in liver, kidney, and adipose tissue have been delineated, and many of the transcription factors that bind to these elements have been identified. Transgenic mice have been especially useful in elucidating the physiological roles of specific sequence elements in the PEPCK-C gene promoter and in demonstrating the key role played at these sites by the isoforms of CAAT/enhancer binding protein in patterning of PEPCK-C gene expression during the perinatal period. The PEPCK-C gene provides a model for the metabolic control of gene transcription.

Animals↗
PubMed1996

Receptor-mediated gene transfer into macrophages.

Gene transfer systems targeting various receptors have been developed to introduce functional genes into cells in culture and into intact animals. A synthetic molecular conjugate, consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor, was constructed and complexed to expression plasmids containing either the Photinus pyralis luciferase or Escherichia coli beta-galactosidase (lacZ) reporter genes. The DNA complexes were used to transfect murine macrophages isolated from peritoneal exudates in vitro. Luciferase and beta-galactosidase activity was found in transfected cells in culture, whereas complexes consisting of an irrelevant plasmid bound to mannosylated polylysine or the expression plasmid bound to galactosylated polylysine resulted in no detectable transgene expression. Gene transfer was inhibited by the addition of excess mannosylated bovine serum albumin to the culture medium before transfection. Reporter genes were also transferred into macrophages residing in the spleen and liver of adult animals using this system. Luciferase activity was maximal at 4 days after transfection and decreased to lower levels by 16 days. Transgene expression conformed to the distribution of cells that had nonspecific esterase, a cytochemical marker for macrophages. Thus, this system can be used to introduce functional genes into macrophages and may be an approach to the treatment of storage diseases that affect the reticuloendothelial system.

Animals↗
PubMed1996

Animal models for studying the genetic basis of metabolic regulation.

Modern genetics has developed methods to modify the expression of genes in animals to study the factors responsible for the tissue-specific expression and hormonal and dietary regulation of metabolic processes. As these methods are applied to genes that code for critical proteins in metabolic pathways, a new insight into the control of metabolism is emerging. There are three general approaches currently in use. First, is the introduction of genes into the germ line to create transgenic animal models in which the gene of interest is over-expressed. This model is of particular value for promoter analysis because it is possible to introduce specific mutations into a putative regulatory region of a transgene and study its transcriptional control in the intact animal. Second, the developmental function of a gene product and its effect on various metabolic processes in a mouse can be directly determined by deleting a gene of interest by homologous recombination. Gene "knockout" mice are currently available with deletions in the genes for a variety of transcription factors and other biologically active proteins, permitting a critical analysis of the proteins responsible for the metabolic patterning of the animal. Third, the metabolic role of a gene of interest in a specific tissue can be studied by ablating its mRNA by the introduction of a transgene that codes for antisense mRNA targeted against the gene transcript. Because it is possible to use a transgene with a tissue-specific promoter, this procedure allows the isolation of the metabolic effect to a selected tissue in the transgenic animal. Taken together, these procedures provide a unique set of metabolic models for an in-depth study of metabolic regulation. This review will present examples of selected animal models currently available and will outline the challenge these animals present for investigators in the nutritional sciences.

Animals↗
PubMed1995

The cyclic AMP response elements of the genes for angiotensin converting enzyme and phosphoenolpyruvate carboxykinase (GTP) can mediate transcriptional activation by CREM tau and CREM alpha.

The potential of the CREM family of proteins to activate transcription of the genes encoding the testis-specific isozyme of angiotensin converting enzyme (ACET) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (EC 4.1.1.32) were investigated. Both CREM tau and CREM alpha bind efficiently to the putative cyclic AMP response element (CRE) present in the ACET gene (CRET) and to the CRE in the PEPCK gene. In HepG2 cells, the CRE was required for the strong stimulation by CREM tau of the expression of a chimeric PEPCK (-210 to +73)-chloramphenicol acetyl transferase (CAT) gene. The CRE could be mutated to the CRET sequence without losing the stimulatory effects of CREM tau. However, a similar chimeric gene driven by the regulatory region of the ACET gene, which contains the CRET site, could only be stimulated by CREM tau when its imperfect TATA element was mutated to an authentic TATA. Surprisingly, CREM alpha, an alleged inhibitor of CRE-mediated transcription, stimulated the expression of both PEPCK-CAT and ACET-CAT genes in HepG2 cells, a process which required the presence of the CRE and the CRET sites, respectively. In contrast, when the same CRE elements were used to drive the transcription of a chimeric gene containing the thymidine kinase promoter linked to the CAT structural gene, CREM alpha inhibited its expression in HepG2 and JEG3 cells. The expression of the same chimeric gene, however, was stimulated by CREM alpha in F9 embryonal carcinoma cells. These results demonstrated that the nature of the transcriptional effects of CREM isoforms on CRE-mediated transcription depends on the specific gene, the specific cell type and the promoter context of the CRE site.

Animals↗
PubMed1995

C/EBP alpha: a critical regulator of genes governing integrative metabolic processes.

The role of C/EBPalpha in the developmental expression of a subset of genes governing essential metabolic processes has recently been elucidated using a mutant mouse model that lacks this transcription factor. The mutation results in a failure of the liver and white and brown adipose tissue to develop normal metabolic functions in the immediate perinatal period, including hepatic glycogen synthesis and gluconeogenesis and the synthesis and deposition of triglyceride in adipose tissue. The metabolic alterations are very similar to those of human infants born prior to the third trimester and suggest that many of the medical complications of prematurity are a result of the lack of activation of C/EBPalpha in development.

Adipose Tissue, Brown↗
PubMed1995

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

Animals↗
PubMed1994

An evaluation of receptor-mediated gene transfer using synthetic DNA-ligand complexes.

Receptor-mediated gene transfer is an attractive method for therapeutically correcting human genetic diseases since it permits the targeting of DNA to cellular receptors in specific tissues of adult animals. Genes introduced by this technique have been shown to be expressed in the target tissue for varying periods. However, to be useful for gene therapy, it is critical that both the chemical properties and physical interactions of the reagents involved in the design of the DNA delivery vehicle be rigorously characterized. In this review, we discuss the critical steps in the preparation of the DNA-ligand complex and the factors involved in the delivery and regulated expression of a transgene in animal tissues. The feasibility of using this technique for the therapeutic delivery of genes to mammalian tissues will also be evaluated.

Animals↗
PubMed1994

Gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake.

Receptor-mediated gene transfer has been used to introduce genes into tissues of animals in vivo. The genes introduced by this approach have been transiently expressed at low levels in animal tissues. High levels of expression, for longer periods, have been attained by the induction of cell division (i.e., partial hepatectomy) or disruption of lysosomal degradation of the DNA. We have studied the correlation of specific structural features on the DNA/ligand complexes with their ability to efficiently introduce DNA into the livers of intact animals. A chimeric gene containing the phosphoenolpyruvate carboxykinase gene promoter (nucleotides -460 to +73) linked to the structural gene for human factor IX (PEPCK-hFIX gene) was condensed with galactosylated poly(L-lysine) by titration with NaCl, resulting in complexes of defined size (10-12 nm in diameter) and shape. The PEPCK-hFIX gene complex was injected into the caudal vena cava of adult rats and the conjugated DNA was specifically targeted to the livers of the animals; no detectable DNA was noted in other tissues. The plasmid containing the PEPCK-hFIX gene was found as an episome in the livers of the rats 32 days after injection of the DNA complex. Human factor IX DNA, mRNA, and functional protein were detected up to 140 days after administration of the DNA complex (the duration of the experiment). Transcription from the PEPCK promoter could be induced over the entire course of the experiment by feeding the rats a high-protein, carbohydrate-free diet. We conclude that the structure of the DNA/ligand complexes is of key importance for the successful introduction of genes into the tissues of animals by receptor-mediated endocytosis.

Animals↗
PubMed1994

An analysis of regulatory elements in the phosphoenolpyruvate carboxykinase (GTP) gene which are responsible for its tissue-specific expression and metabolic control in transgenic mice.

Sequences in the gene for P-enolpyruvate carboxykinase (PEPCK) which are responsible for its complex pattern of transcriptional control were determined using transgenic mice containing a chimeric PEPCK-bovine growth hormone (bGH) gene consisting of a segment of the PEPCK promoter from -460 to +73, with mutations in specific regulatory domains. A mutation in the cAMP response element (CRE) (-87 to -74), which binds CCATT/enhancer-binding protein beta (C/EBP beta) and/or cAMP response element-binding protein (CREB), resulted in a 4- and 20-fold elevation in the level of bGH mRNA in the liver and kidney of transgenic mice, respectively. Expression of the PEPCK-bGH gene in the liver was reduced 60% by a mutation in the P3 (I) region (-248 to -230), whereas expression in the kidney was increased 10-fold by the same mutation. A mutation in the P2 region (-200 to -164) greatly reduced expression of the PEPCK-bGH gene in the kidney but not in the liver. Induction of hepatic PEPCK-bGH gene expression by Bt2cAMP was eliminated by mutations in the CRE, P1, P3(I), or by a double mutation of the CRE and P3(I). Mutations in the CRE or P3(I) regions of the PEPCK promoter did not interfere with the expected induction of the PEPCK-bGH gene in the liver at birth. None of the mutations in the PEPCK promoter interfered with the induction of transcription of the PEPCK-bGH gene in the liver when mice were fed a carbohydrate-free diet or the deinduction of transcription from the PEPCK promoter caused by a diet high in carbohydrate, whereas a mutation in P2 (an HNF-1 binding domain) eliminated dietary regulation of transcription of the transgene in the kidney. A model to explain the role of the various elements in the PEPCK promoter on the control of PEPCK gene transcription in the liver and kidney is presented.

Animals↗
PubMed1994

Metabolic regulation of gene transcription.

The impact of nutrients on gene expression has become an area of considerable interest as the number of genes coding for key regulatory proteins in metabolic pathways are studied in detail. This has been greatly aided by a number of new techniques developed to study gene transcription in animals. We will use as an example studies on the regulation of transcription of the gene coding for P-enolpyruvate carboxykinase, a key enzyme in hepatic and renal gluconeogenesis. The promoter for P-enolpyruvate carboxykinase contains a number of regulatory elements within 500 bp of the start-site of gene transcription that are required for the response of the gene to metabolic signals. These elements bind tissue-specific transcription factors in complex patterns of interactions, which result in the coordinate control of P-enolpyruvate carboxykinase gene expression. An analysis of the regulation of transcription of this gene involves the use of a number of techniques ranging from gene transfection into cells in culture to the introduction of chimeric genes containing the P-enolpyruvate carboxykinase promoter into transgenic mice. This review presents a progress report on the current status of research on the nutritional and hormonal regulation of transcription of the P-enolpyruvate carboxykinase gene.

Activating Transcription Factor 2↗