The induction of adipose conversion in 3T3-L1 cells by bezafibrate (Brandes, R., Hertz, R. Arad R., Naishtat S., Weil, S. and Bar-Tana, J. (1987) Life Sci., 40, 935-941) was enhanced by ...dibutyryl-cAMP as well as forskolin, theophylline or isobutylmethylxanthine added to the incubation medium together with the bezafibrate inducer. The synergistic effect of bezafibrate and dibutyryl-cAMP resulted in enhancing the expression of late markers of adipose conversion, e.g., lipid accumulation or glycerol-3-phosphate dehydrogenase activity and its mRNA. This enhanced expression of late markers was reflected in shortening the time period required for their first appearance as well as increasing their yield during the course of adipose conversion. By following the accumulation of glutamine synthetase mRNA serving as an early marker for adipose conversion, the synergistic effect of bezafibrate and dibutyryl-cAMP was already evident as early as 5 h following their addition to confluent 3T3-L1 cells. Hence, the induction of adipose conversion by bezafibrate in 3T3-L1 cells appears to involve an early event which is rate-limited by the availability of intracellular cAMP.
The liver is equipped with a repertoire of enzymatic activities essential for executing its specialized role in metabolism, the expression of which is regulated during development. The liver-specific ...phenotype is the consequence of a developmental tissue-specific program of gene expression. Sequences close to many characterized structural liver-specific genes (cis-regulatory elements) regulate their transcription. Identification of such cis-regulatory elements, capable of conferring a hepatocyte-specific gene expression, has been achieved by the introduction of chimeric genes into germ lines, producing transgenic animals, into differentiated cultured cells and into a cell-free transcription system. Such cis-elements in the DNA are recognized by specific DNA-binding nuclear proteins (trans-acting factors) which are liver-enriched and developmentally controlled. The interaction of defined cis-acting elements, near liver-specific genes, with liver-specific trans-acting factors might result in the differentiation of cells of the endoderm lineage into hepatocyte cells.
Recently we have developed a method for direct introduction of calcium phosphate-precipitated DNA into newborn rats. To examine whether the foreign DNA can replicate, a plasmid containing a polyoma ...origin of replication was injected into newborn mice. The plasmid was found intact in liver and spleen and able to transform bacteria. The foreign DNA had disappeared by the seventh day after injection. Yet, the plasmid DNA containing the polyoma origin of replication had undergone replication in both the liver and the spleen.
Rat fetuses of 17-19-day gestation were injected in utero with 5-azacytidine (two to three daily injections of 40 micrograms/fetus). Neonates were injected with seven daily injections (1 mg/kg). DNA ...samples were isolated from the fetal and neonatal livers and neonatal spleen and subjected to analysis of their methylation status. Overall methylation was analyzed by the nearest-neighbor analysis (at CpG sites) and the pattern of methylation at CCGG sites by Southern blot analysis using phosphoenolpyruvate carboxykinase (PEPCK) sequences as probes. While DNAs from the liver and spleen undergo hypomethylation to the same extent in response to the 5-azacytidine treatment, the changes in the methylation patterns of the PEPCK gene in the two tissues are strikingly different. The changes observed indicate that a decrease in the methylase activity (inhibition by 5-azacytidine) results in site- and tissue-specific hypomethylation. The tissue-specific changes in the methylation pattern are associated with a tissue-specific expression of the PEPCK gene. Although the gene is hypomethylated by azacytidine in both liver and spleen, it is expressed only in the liver. The expression of already active genes (PEPCK in the kidney and albumin in the liver) is not further enhanced by the drug.
To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-lclc7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by ...appreciably expressing the α-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1α (HNF-1α) and HNF-4 and the late-expressed factor C/ΕΒΡα, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1α but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1α resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.
A sequential pattern of interactions of trans-acting factors in rat liver with the phosphoenolpyruvate carboxykinase promoter during late development was observed. A liver-enriched factor, possibly ...AF1, interacted with the promoter in fetal liver, whereas a factor with the characteristics of C/EBP bound the promoter after birth with the onset of the gene expression.
To study the liver-specific trans activation of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene, the PEPCK promoter was linked to a reporter gene and was microinjected into Xenopus laevis ...oocytes alone or in conjunction with rat liver poly(A)
+
RNA. The rat liver mRNA markedly enhanced the expression of the PEPCK-chimeric construct. This effect appeared to be sequence specific, as it was dependent on the presence of the intact promoter. Moreover, the RNA effect was limited to mRNA preparations from PEPCK-expressing tissues only. Finally, microinjection of size-fractionated liver mRNA revealed that the trans-acting factor(s) is encoded by RNA of 1,600 to 2,000 nucleotides, providing a direct bioassay for the gene(s) involved in this tissue-specific trans-activation process.
Structural conservation of cytosolic phospho
enol
pyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species ...tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phospho
enol
pyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phospho
enol
pyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.
Structural conservation of cytosolic phospho
enolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species ...tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phospho
enolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phospho
enolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.
Structural conservation of cytosolic phosphoenolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species ...tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phosphoenolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phosphoenolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.
