RESUMO
AIMS/HYPOTHESIS: Glucokinase plays a key role in glucose homeostasis and the expression of its gene is differentially regulated in pancreatic beta cells and in the liver through distinct promoters. The factors that determine the tissue-specific expression of the glucokinase gene are not known. Putative binding sites for hepatocyte nuclear factor (HNF)-6, the prototype of the ONECUT family of transcription factors, are present in the hepatic promoter of the glucokinase gene and hnf6 knockout mice are diabetic [corrected]. We hypothesized that HNF-6 controls the activity of the hepatic glucokinase promoter. METHODS: We tested the binding of recombinant HNF-6 to DNA sequences from the mouse hepatic glucokinase promoter in vitro and the effect of HNF-6 on promoter activity in transfected cells. We investigated in vivo the role of HNF-6 in mice by examining the effect of inactivating the hnf6 gene on glucokinase gene-specific deoxyribonuclease I hypersensitive sites in liver chromatin and on liver glucokinase mRNA concentration. RESULTS: HNF-6 bound to the hepatic promoter of the glucokinase gene and stimulated its activity. Inactivation of the hnf6 gene did not modify the pattern of deoxyribonuclease I hypersensitive sites but was associated with a decrease of liver glucokinase mRNA to half the control value. CONCLUSIONS/INTERPRETATION: Although HNF-6 is not required to open chromatin of the hepatic promoter of the glucokinase gene, it stimulates transcription of the glucokinase gene in the liver. This could partly explain the diabetes observed in hnf6 knockout mice.
Assuntos
Regulação da Expressão Gênica/fisiologia , Glucoquinase/genética , Proteínas de Homeodomínio/fisiologia , Fígado/fisiologia , Transativadores/fisiologia , Animais , Cromatina/fisiologia , Desoxirribonuclease I/fisiologia , Expressão Gênica/efeitos dos fármacos , Glucoquinase/metabolismo , Fator 6 Nuclear de Hepatócito , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/farmacologia , Fígado/metabolismo , Camundongos , Camundongos Knockout/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/antagonistas & inibidores , Ratos , Proteínas Recombinantes/metabolismo , Transativadores/genética , Transativadores/farmacologia , Células Tumorais CultivadasRESUMO
This brief review focuses on the transcriptional regulation of liver carnitine palmitoyltransferase I (L-CPT I) by pancreatic and thyroid hormones and by long-chain fatty acids (LCFA). Both glucagon and 3,3',5-tri-iodothyronine (T(3)) enhanced the transcription of the gene encoding L-CPT I, whereas insulin had the opposite effect. Interestingly, the transcriptional effect of T(3) required, in addition to the thyroid-responsive element, the co-operation of a sequence located in the first intron of L-CPT I gene. Non-esterified fatty acids rather than acyl-CoA ester or intra-mitochondrial metabolite were responsible for the transcriptional effect on the gene encoding L-CPT I. It was shown that LCFA and peroxisome proliferators stimulated L-CPT I gene transcription by distinct mechanisms. Peroxisome proliferator stimulated L-CPT I gene transcription through a peroxisome-proliferator-responsive element (PPRE) located at -2846 bp, whereas LCFA induced L-CPT I gene transcription through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent mechanism owing to a sequence located in the first intron of the gene.
Assuntos
Carnitina O-Palmitoiltransferase/genética , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica , Hormônios/metabolismo , Fígado/enzimologia , Animais , Carnitina O-Palmitoiltransferase/biossíntese , Ácidos Graxos/química , Insulina/metabolismo , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Liver carnitine palmitoyltransferase I (L-CPT I) catalyses the transfer of long-chain fatty acid (LCFA) for translocation across the mitochondrial membrane. Expression of the L-CPT I gene is induced by LCFAs as well as by lipid-lowering compounds such as clofibrate. Previous studies have suggested that the peroxisome-proliferator-activated receptor alpha (PPARalpha) is a common mediator of the transcriptional effects of LCFA and clofibrate. We found that free LCFAs rather than acyl-CoA esters are the signal metabolites responsible for the stimulation of L-CPT I gene expression. Using primary culture of hepatocytes we found that LCFAs failed to stimulate L-CPT I gene expression both in wild-type and PPARalpha-null mice. These results suggest that the PPARalpha-knockout mouse does not represent a suitable model for the regulation of L-CPT I gene expression by LCFAs in the liver. Finally, we determined that clofibrate stimulates L-CPT I through a classical direct repeat 1 (DR1) motif in the promoter of the L-CPT I gene while LCFAs induce L-CPT I via elements in the first intron of the gene. Our results demonstrate that LCFAs can regulate gene expression through PPARalpha-independent pathways and suggest that the regulation of gene expression by dietary lipids is more complex than previously proposed.
Assuntos
Carnitina O-Palmitoiltransferase/genética , Ácidos Graxos/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Fígado/enzimologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Clofibrato/farmacologia , Primers do DNA , Inibidores Enzimáticos/farmacologia , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Stat proteins are SH2 domain-containing transcription factors that are activated by various cytokines and growth factors. In a previous work, we have identified Stat 5B as a substrate of the insulin receptor based on yeast two-hybrid and mammalian cell transfection studies. In the present study, we have approached the biological relevance of the interaction between the insulin receptor and the transcription factor Stat 5B. Firstly, we show that both insulin and insulin-like growth factor I lead to tyrosine phosphorylation of Stat 5B, and this promotes binding of the transcription factor to the beta-casein promoter containing a Stat 5 binding site. Further, we demonstrate that insulin stimulates the transcriptional activity of Stat 5B. Activation of Stat 5B by insulin appears to be Jak2-independent, whereas Jak2 is required for GH-induced Stat 5B activation. Hence the pathway by which Stat 5B is activated by insulin is different from that used by GH. In addition, by using Jak1- and Tyk2-deficient cells we exclude the involvement of both Jak1 and Tyk2 in Stat 5B activation by insulin. Taken together, our results strengthen the notion that insulin receptor can directly activate Stat 5B. More importantly, we have identified a Stat 5 binding site in the human hepatic glucokinase promoter, and we show that insulin leads to a Stat 5B-dependent increase in transcription of a reporter gene carrying this promoter. These observations favor the idea that Stat 5B plays a role in mediating the expression of the glucokinase gene induced by insulin. As a whole, our results provide evidence for the occurrence of a newly identified circuit in insulin signaling in which the cell surface receptor is directly linked to nuclear events through a transcription factor. Further, we have revealed an insulin target gene whose expression is, at least in part, dependent on Stat 5B activation and/or binding.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Glucoquinase/genética , Insulina/farmacologia , Proteínas do Leite , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Transativadores/fisiologia , Transcrição Gênica , Células 3T3 , Animais , Carcinoma Hepatocelular/metabolismo , DNA/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Janus Quinase 1 , Janus Quinase 2 , Fígado/enzimologia , Neoplasias Hepáticas/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fosfotirosina/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT5 , Transfecção , Células Tumorais CultivadasRESUMO
A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.
Assuntos
Regiões 5' não Traduzidas/genética , Regiões Promotoras Genéticas/genética , Receptores de Glucagon/genética , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Éxons/genética , Feminino , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Íntrons/genética , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação/genética , Ratos , Ratos Wistar , Elementos de Resposta/genética , Alinhamento de Sequência , Fator de Transcrição Sp1/fisiologia , Transcrição Gênica/genética , TransfecçãoRESUMO
We have recently cloned the murine glucagon receptor (GR) gene and shown that it is expressed mainly in liver. In this organ, the glucagon-GR system is involved in the control of glucose metabolism as it initiates a cascade of events leading to release of glucose into the blood stream, which is a main feature in several physiological and pathological conditions. To better define the metabolic regulators of GR expression in liver we analyzed GR mRNA concentration in physiological conditions associating various glucose metabolic pathways in vivo and in vitro in the rat and in the mouse. First, we report that the concentration of the GR mRNA progressively increased from the first day of life to the adult stage. This effect was abolished when newborn rodents were fasted. Second, under conditions where intrahepatic glucose metabolism was active such as during fasting, diabetes, and hyperglycemic clamp, the concentration of GR mRNA increased independent of the origin of the pathway that generated the glucose flux. These effects were blunted when hyperglycemia was corrected by phlorizin treatment of diabetic rats or not sustained during euglycemic clamp. In accordance with these observations, we demonstrated that the glycolytic substrates glucose, mannose, and fructose, as well as the gluconeognic substrates glycerol and dihydroxyacetone, increased the concentration of GR mRNA in primary cultures of hepatocytes from fed rats. Glucagon blunted the effect of glucose without being dominant. The stimulatory effect of those substrates was not mimicked by the nonmetabolizable carbohydrate L-glucose or the glucokinase inhibitor glucosamine or when hepatocytes were isolated from starved rats. In addition, inhibitors of gluconeogenesis and lipolysis could decrease the concentration of GR mRNA from hepatocytes of starved rats. Combined, these data strongly suggest that glucose flux in the glycolytic and gluconeogenic pathways at the level of triose intermediates could control expression of GR mRNA and participate in controlling its own metabolism.
Assuntos
Glucose/metabolismo , Fígado/metabolismo , RNA Mensageiro/biossíntese , Receptores de Glucagon/biossíntese , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos CBA , Ratos , Receptores de Glucagon/genéticaRESUMO
The Gk activity locus affects expression of hepatic glucokinase (GK) in mice. Analysis of microsatellites within the mouse GK gene locus revealed two major haplotypes in 19 of 22 inbred strains predictive of either high or low hepatic GK gene expression. C3H/HeJ mice, a high-activity strain, and two other wild-derived strains contain less common haplotypes. No coding sequence differences were found in hepatic GK-coding sequences from representative high and low Gk activity strains, thereby excluding kinetic abnormalities as the basis for hepatic GK activity differences. Screening of approximately 10 kb of potential regulatory DNA, including all eight known and three of four newly identified DNase I-hypersensitive sites, by restriction enzyme fingerprinting-single-strand conformation analysis revealed a tetranucleotide microsatellite, the length of which was also predictive of the Gk activity phenotype. This tetranucleotide repeat is located in the first intron of the hepatic transcription unit and lies close to a newly identified liver-specific DNase I-hypersensitive site. These results indicate that the Gk activity alleles are a regulational locus associated with the GK gene locus.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucoquinase/genética , Fígado/enzimologia , Animais , Haplótipos , Camundongos , Repetições de Microssatélites , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
Glucokinase (EC 2.7.1.2) first appears in rat liver two weeks after birth and increases rapidly after weaning on to a high-carbohydrate diet. We investigated the role of triiodothyronine and retinoic acid in the absence of insulin on the first expression of the glucokinase gene in primary cultures of hepatocytes from 10 day-old rats. These two hormones were able to induce a rapid accumulation of liver glucokinase mRNA, secondarily to a stimulation of gene transcription during the first 24 h of culture. Moreover, the effects of individual hormones were not additive. Finally, glucokinase mRNA stability was not modified by these hormones. This suggests that triiodothyronine and retinoic acid act on glucokinase gene at the transcriptional.
Assuntos
Glucoquinase/genética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Tretinoína/farmacologia , Tri-Iodotironina/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Relação Dose-Resposta a Droga , Feto/enzimologia , Feto/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Cinética , Fígado/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Tretinoína/administração & dosagem , Tri-Iodotironina/administração & dosagemRESUMO
Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1-day-old newborn rats, but not in cultured hepatocytes from 21-day-old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver-specific DNase I hypersensitive sites located in the 5' flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site-1 and hypersensitive site-2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1-day-old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase-I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site-1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase-I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin-response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucoquinase/genética , Insulina/farmacologia , Fígado/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico , Animais , Animais Recém-Nascidos , Sequência de Bases , Pegada de DNA , Desoxirribonuclease I/metabolismo , Embrião de Mamíferos/citologia , Indução Enzimática , Glucoquinase/biossíntese , Fígado/citologia , Fígado/enzimologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Simplexvirus/genética , Timidina Quinase/genéticaRESUMO
We cloned and characterized an 83-kb fragment of mouse genomic DNA containing the entire glucokinase (GK) gene. The 11 exons of the gene span a total distance of 49 kb, with exons 1 beta and 1L being separated by 35 kb. A total of 25,266 bp of DNA sequence information was determined: from approximately -9.2 to approximately +15 kb (24,195 bp), relative to the hepatocyte transcription start site, and from -335 to +736 bp (1071 bp), relative to the transcription start site in beta cells. These sequences revealed that mouse GK is > 94% identical to rat and human GK. Mouse hepatic GK mRNA is regulated by fasting and refeeding, as also occurs in the rat. Alignment of the upstream and downstream promoter regions of the mouse, rat, and human genes revealed several evolutionarily conserved regions that may contribute to transcriptional regulation. However, fusion gene studies in transgenic mice indicate that the conserved regions near the transcription start site in hepatocytes are themselves not sufficient for position-independent expression in liver. Analysis of the chromatin structure of a 48-kb region of the mouse gene using DNase I revealed eight liver-specific hypersensitive sites whose locations ranged from 0.1 to 36 kb upstream of the liver transcription start site. The availability of a single, contiguous DNA fragment containing the entire mouse GK gene should allow further studies of cell-specific expression of GK to be performed.
Assuntos
Evolução Biológica , Mapeamento Cromossômico , Glucoquinase/genética , Camundongos/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA/química , DNA/genética , Primers do DNA , Desoxirribonuclease I , Éxons , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Glucoquinase/biossíntese , Humanos , Fígado/enzimologia , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The initial accumulation of glucokinase mRNA in response to insulin in cultured hepatocytes from 10-day-old suckling rats was characterized by a delay of 18-24 h with a maximal level reached after 48 h. This delay is not observed in cultured adult rat hepatocytes. When hepatocytes from 10-day-old suckling rats were cultured for 48 h in the presence of insulin (to obtain a maximal accumulation of glucokinase mRNA) and then deprived of insulin for 18 h, glucokinase mRNA returned to very low levels. Reexposure of these cultured hepatocytes to insulin allowed a rapid accumulation of glucokinase mRNA, with a maximal level reached after 8 h, as in adult rat hepatocytes. The aim of the present study was to investigate the factors responsible for the delay in insulin action during first exposure to insulin. The difference in the kinetics of glucokinase mRNA accumulation after the first and secondary exposure to insulin was due to differences in the rate of transcriptional activity of the glucokinase gene, as shown by a run-on assay on isolated nuclei. The half-life of glucokinase mRNA was similar after the first and second exposure to insulin. The delay in the initial accumulation of glucokinase mRNA in response to the first exposure to insulin was not due to elevated levels of cAMP (a potent inhibitor of glucokinase gene expression) or to a defect in insulin signalling (insulin inhibited without delay phosphoenolpyruvate carboxykinase gene expression). In contrast, it was markedly dependent upon whether glucokinase has been already expressed in vivo. Hepatocytes from rats that had already expressed glucokinase in vivo (suckling rats force-fed with glucose or rats weaned to a high-carbohydrate diet) showed no delay in their response to insulin in culture, whereas hepatocytes from rats that have never expressed glucokinase in vivo (suckling rats or rats weaned to a high-fat diet) showed a delay of 24 h. Two different inhibitors of protein synthesis (cycloheximide and puromycin) prevented the initial accumulation of glucokinase mRNA in response to the first exposure to insulin but not to the secondary accumulation of glucokinase mRNA in response to reexposure to insulin. This suggests that the synthesis of one or several insulin-dependent proteins is necessary for the first activation of glucokinase gene transcription in response to the first exposure to insulin.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucoquinase/genética , Insulina/farmacologia , Fígado/enzimologia , Biossíntese de Proteínas , Envelhecimento/metabolismo , Animais , Animais Lactentes , Células Cultivadas , AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Glucagon/farmacologia , Insulina/sangue , Fígado/citologia , Fígado/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Glucokinase first appears in the liver of the rat 2 weeks after birth and its activity rapidly increases after weaning on to a high-carbohydrate diet. The appearance of glucokinase is principally due to the increase of plasma insulin and to the decrease of plasma glucagon concentrations. Oral glucose administration to 1- or 10-day-old suckling rats induced an increase in plasma insulin and a fall in plasma glucagon and allowed a rapid accumulation of liver glucokinase mRNA, secondarily to a stimulation of gene transcription. When unrestrained late pregnant rats were infused with glucose during 36 h to induce an increase in fetal plasma insulin and a decrease in fetal plasma glucagon concentrations, glucokinase mRNA was detectable in fetal liver but the level was 100-fold lower than that observed in 1- or 10-day-old suckling rats. It is suggested that the hormonal environment did not allow glucokinase gene expression to be induced in fetal liver and that the absence of expression of glucokinase in suckling rat liver is due to the presence of low plasma insulin and high plasma glucagon levels. The chromatin structure of the glucokinase gene was examined during development by identification of DNase-I-hypersensitive sites from the region comprised between -8 kb upstream and +4 kb downstream of the cap site. Five hypersensitive sites were found: four liver-specific sites upstream of the cap site and one non-specific site in the first intron. These sites are already present in term fetus but the intensity of the two proximal sites located upstream of the cap site increase markedly after birth. This suggests that these sites could be implicated in the regulation of glucokinase gene expression by insulin and glucagon. Full DNase-I-hypersensitivity of these two proximal sites seems necessary for the mature response of glucokinase gene in response to changes in pancreatic hormones concentrations.
Assuntos
Glucoquinase/biossíntese , Glucose/farmacologia , Fígado/enzimologia , Animais , Animais Recém-Nascidos , Cromatina , Desoxirribonuclease I/metabolismo , Indução Enzimática/efeitos dos fármacos , Feminino , Feto/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucagon/sangue , Glucoquinase/genética , Insulina/sangue , Fígado/efeitos dos fármacos , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transcrição GênicaRESUMO
Cultured adult rodent hepatocytes are extensively used as a model system for gene transfer in vitro. In the present study, we examined the influence differentiation status and growth capacity of the hepatocytes on their infectivity in vitro by a retroviral vector. These parameters were initially studied in primary cultures of rat hepatocytes transduced with an ecotropic retroviral vector containing Escherichia coli beta-galactosidase. However, significant differences observed in the infectivity of hepatocytes from 12-day-old and adult rats led us to also examine hepatocytes from a transgenic mouse strain in which the SV40 large T antigen is fused to the regulatory sequences of the human anti-thrombin III gene. The large T antigen is expressed in the liver and these mice develop hepatoma within 7 months. A comparison of infectivity of hepatocytes from normal and transgenic mice of different ages indicated that in contrast to previous reports, hepatocytes which express differentiated functions during the first week of culture can still be efficiently infected by retroviral vectors. Optimal infection was observed between the second and fourth day of culture and does not appear to be due to transient cell dedifferentiation, but is more likely due to transient mitotic activity of mice cells since the role of growth factors seems crucial for infection. The peak of infection did not appear to correspond to transient cell dedifferentiation. We also found differences of infectivity between hepatocytes from normal and transgenic mice of different ages. Such differences are correlated with differences in in vitro BrdU incorporation, which was used to determine the proportion of dividing hepatocytes. These results indicate that the efficiency of infectivity of hepatocytes by recombinant retrovirus is probably related to their normal proliferative potential and not to some dedifferentiated stage. Hence these findings provide a model for efficient gene transfer in differentiated cells and suggest an approach for studies of liver-specific gene regulation and for somatic gene therapy of metabolic diseases as well.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Neoplasias Hepáticas Experimentais/genética , Retroviridae/genética , Transformação Genética , Animais , Células Cultivadas/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Vetores Genéticos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Ratos , Ratos Wistar , Infecções por Retroviridae , alfa-Fetoproteínas/análiseRESUMO
In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.
Assuntos
Isoenzimas/genética , Fígado/enzimologia , Piruvato Quinase/genética , RNA Mensageiro/biossíntese , Vanadatos/farmacologia , Animais , Northern Blotting , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Glucoquinase/genética , Glucose/farmacologia , Insulina/farmacologia , Fígado/efeitos dos fármacos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Endogâmicos , Tionucleotídeos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.
Assuntos
Frutose-Bifosfato Aldolase/genética , Íntrons , Fígado/enzimologia , Regiões Promotoras Genéticas , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/genética , Sequência Consenso , Desoxirribonuclease I , Fibroblastos/enzimologia , Frutose-Bifosfato Aldolase/biossíntese , Humanos , Sequências Reguladoras de Ácido Nucleico , Sensibilidade e Especificidade , Transfecção , Células Tumorais CultivadasRESUMO
Insulin action on gene expression can be glucose-dependent or -independent. Accumulation of aldolase B mRNA and of an unidentified 5.4-kilobase mRNA as well as accumulation of L-type pyruvate kinase mRNAs (Decaux, J.F., Antoine, B., and Kahn, A. (1989) J. Biol. Chem. 264, 11584-11590) in cultured hepatocytes isolated from fasted rats require the presence of both glucose and insulin, these agents not being effective individually. In contrast, maintaining the amount of albumin and transferrin mRNAs in these hepatocytes requires the presence of insulin alone, glucose having no effect by itself. Transcription of the albumin gene, investigated by run-on assay, is active in the presence of insulin alone, with or without glucose, whereas transcription of the aldolase B gene is stimulated by glucose and insulin together, but not by insulin or glucose alone. In addition, the stability of the albumin and aldolase B mRNAs in cultured hepatocytes is lowered in the absence of glucose and insulin together as compared to the stability in the presence of one or both agents. These results confirm that transduction of the insulin signal occurs via distinct pathways; one of these pathways could involve a secondary insulin-dependent modification of metabolite concentration, whereas other pathways could be more directly related to the activity(ies) of the occupied insulin receptor.
Assuntos
Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Insulina/farmacologia , Albuminas/genética , Animais , Northern Blotting , Células Cultivadas , DNA/genética , Frutose-Bifosfato Aldolase/genética , Cinética , Fígado/citologia , Fígado/efeitos dos fármacos , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , RNA Mensageiro/química , Ratos , Transcrição Gênica/efeitos dos fármacos , Transferrina/genéticaRESUMO
L-type pyruvate kinase gene regulation is an excellent model of gene control by hormones and diet. In vivo and ex vivo experiments allowed us to established that thyroid hormones and glucocorticoids act on pyruvate kinase gene expression at the post-transcriptional level. In contrast, glucose and insulin together stimulate transcription of this gene while glucagon inhibits it. Insulin or glucose are individually inefficient and glucagon-dependent transcriptional inhibition seems to be dominant in insulin + glucose-dependent activation. A 14-kbp fragment encompassing the entire pyruvate kinase gene and 3.2-kbp of 5' flanking sequences is expressed in transgenic mice exactly like the endogenous gene; the 3.2-kbp upstream region is sufficient to confer this tissue-specific and hormone/diet-regulated expression to reporter genes. In vivo, DNAse I hypersensitivity analysis revealed the presence of 3 liver-specific groups of hypersensitive sites (HSS). The proximal sites, between + 1 and -183 bp with respect to the start site of transcription, were, in addition, transcription-dependent. The nature and functional role of proteins binding to this proximal upstream sequence were analyzed by in vitro binding and cell free transcription experiments. The existence of more upstream cis-acting elements was investigated by transient transfection assays using differentiated hepatoma cell lines and hepatocytes in primary culture. These experiments permitted the detection of an extinguisher active in hepatoma Hep G2 cells but not in hepatocytes, and of an activating element which could correspond to a distal HSS. Unfortunately, this investigation has not yet allowed us to determine with accuracy the DNA elements responsible for response to diet and hormones.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucagon/fisiologia , Insulina/fisiologia , Piruvato Quinase/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Ingestão de Alimentos/fisiologia , Jejum/fisiologia , Humanos , Sequências Reguladoras de Ácido NucleicoRESUMO
During the suckling period, the rats are fed continuously with milk, which is a high-fat low-carbohydrate diet (HF). At weaning, milk is progressively replaced by the rat's laboratory chow which is a high-carbohydrate low-fat diet (HCHO), and this is accompanied by large hormonal modifications: an increase in plasma insulin and a decrease in plasma glucagon concentrations, and by marked changes in metabolic pathways in liver: decrease in hepatic gluconeogenesis and increase in glycolysis and lipogenesis. Most of the data concerning these changes are related to maximal activity of enzymes. The recent availability of specific cDNA probes for phosphoenolpyruvate carboxykinase (PEPCK), and glucokinase (GK) has allowed the study of the role of pancreatic hormones and nutrition in the changes of the expression of these genes at weaning in the rat. Regarding phosphoenolpyruvate carboxykinase gene transcription, the concentration of mRNA as well as the activity of PEPCK are elevated in the liver of suckling rat until the onset of weaning, 21 d after delivery. After weaning to a HCHO diet, both mRNA and activity of PEPCK rapidly decrease to a very low level. In contrast, weaning on an HF diet, which maintains high plasma glucagon and low plasma insulin levels, does not decrease in plasma glucagon concentration and a 90% decrease in PEPCK gene transcription and PEPCK mRNA concentration in 1 h. Regarding glucokinase gene transcription, the concentration of mRNA as well as the activity of GK are not detectable before 15 d after birth in the liver of the rat. They markedly increase when the newborn are weaned on an HCHO diet but not when they are weaned on an HF diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucoquinase/genética , Fígado/enzimologia , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Animais , Glucagon/sangue , Glucoquinase/metabolismo , Insulina/sangue , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Ratos , Transcrição Gênica , DesmameRESUMO
Hepatocytes isolated from adult fasted rats and cultured in the presence of thyroid hormones, glucocorticoids, and in a serum-free medium conserve the essentials of their differentiated function and hormonal sensitivity for at least 1 week. In these cells, the gene for L-type pyruvate kinase is expressed only when glucose and insulin are present together, each of them being inactive by itself. Inhibition of the expression of the L-type pyruvate kinase gene which occurs when glucose and/or insulin are removed from the culture medium is not associated with accumulation of the phosphoenolpyruvate carboxykinase mRNA, which argues against the involvement of intracellular cyclic AMP in this phenomenon. Rather, a transcriptional activator, derived from carbohydrate metabolism and accumulating in the presence of insulin, seems to be needed to support the expression of the L-type pyruvate kinase gene. Glucagon, in vitro as in vivo, inhibits production of the L-type pyruvate kinase mRNAs. In addition to their roles on the production of these mRNAs, glucose and insulin on the one hand and glucagon on the other have profound effects on the stability of the L-type pyruvate kinase messengers: the half-life of the mRNA whose production has been blocked by actinomycin D is 1 h in the presence of glucagon and 24 h in the presence of glucose and insulin. Glucagon and glucose/insulin partially antagonize each other's effect on mRNA stability.