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In vivo, feeding stimulates and starvation inhibits transcription of the malic enzyme gene. In chick-embryo hepatocytes in culture, triiodothyronine (T3) stimulates and glucagon inhibits transcription of this gene. As a first step in the characterization of the involved regulatory mechanisms, fragments of genomic DNA spanning the structural and 5'-flanking regions of the chicken malic enzyme gene were cloned. The coding region of the gene is organized into 14 exons and 13 introns and is greater than 106 kb in length. The size of the gene, the number and lengths of the exons, and positions at which introns are inserted into the coding regions are virtually identical in the chicken and rat genes. When transiently transfected into chick-embryo hepatocytes, 5800 bp of 5'-flanking DNA conferred T3 responsiveness to a linked chloramphenicol acetyltransferase (CAT) reporter gene. Using deletion and site-specific mutations of 5'-flanking DNA, we identified a complex T3 response unit that contains one major T3 response element (T3RE) and several minor ones. The major element contains two degenerate copies of the hexamer, RGGWMA, separated by 4 bp and was a strong repressor in the absence of ligand. Endogenous levels of T3 receptor are sufficient to allow the T3 response elements in the upstream region of the malic enzyme gene to confer responsiveness to T3, suggesting that they are physiologically relevant.

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We have provided a historical and personal description of the analysis of physiological and molecular mechanisms by which diet and hormones regulate the activity of hepatic malic enzyme. For the most part, our analyses have been reductionist in approach, striving for increasingly simpler systems in which we can ask more direct questions about the molecular nature of the signaling pathways that regulate the activity of malic enzyme. The reductionist approaches that were so successful at analyzing molecular mechanisms in cells in culture may now provide the means to analyze more definitively questions about the physiological mechanisms involved in nutritional regulation of gene expression. In addition to physiological questions, however, there are still many aspects of the molecular mechanisms that have not been elucidated. Despite considerable effort from many laboratories, the molecular mechanisms by which T3 regulates transcription are not clear. Similarly, the molecular details for the mechanisms by which glucagon, insulin, glucocorticoids, and fatty acids regulate gene expression remain to be determined. The role of fatty acids is particularly interesting because it may provide a model for mechanisms by which genes are regulated by metabolic intermediates; this is a form of transcriptional regulation widely used by prokaryotic organisms and extensively analyzed in prokaryotic systems, but poorly understood in higher eukaryotes. At any specific time, there is, of course, only one rate of transcription for each copy of the malic-enzyme gene in a cell. Our long-term objective is to understand how signals from all of the relevant regulatory pathways are integrated to bring about that rate.

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The catalytic (C) subunit of cAMP-dependent protein kinase is inhibited by the regulatory (R) subunit and by a thermostable inhibitor (PKI). Both inhibitors also affect the intracellular distribution of the C subunit. Whether injected into the cytoplasm or into the nucleus, free C subunit can enter and exit the nucleus freely. After 30 min its distribution is identical and is independent of the initial site of injection. In contrast, when C is injected into the cytoplasm complexed with R or PKI, the complexes are restricted to the cytoplasm (1-3). However, unlike the R subunit, which is restricted to the cytoplasm like the holoenzyme, free PKI enters the nucleus rapidly following its injection into the cytoplasm. When holoenzyme is injected directly into the nucleus, it cannot exit and return to the cytoplasm. In contrast, nuclear injection of a C.PKI complex results in the rapid exit of the C subunit from the nucleus. In equilibrated cells previously injected with the C subunit, subsequent cytoplasmic injection of either PKI or type 1 R depletes the nucleus of C although PKI does so faster, consistent with its ability to enter the nucleus. Both inhibitors block the cAMP response element-regulated gene expression. Hence PKI may serve as a nuclear scavenger of C providing a mechanism not only for inhibition but also for subcellular localization in the presence of cAMP by restricting the access of the C subunit to the nucleus.

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cAMP-dependent protein kinase mediates a variety of cellular responses in most eukaryotic cells. Many of these responses are cytoplasmic, whereas others appear to require nuclear localization of the catalytic subunit. In order to understand further the molecular basis for subcellular localization of the catalytic subunit, the effect of the heat stable protein kinase inhibitor (PKI) was investigated. The subcellular localization of the catalytic (C) subunit was determined both in the presence and absence of PKI, by microinjecting fluorescently labeled C subunit into single living cells. When injected alone, a significant fraction of the dissociated C subunit localized to the nucleus. When coin-injected with an excess of PKI, little of the C subunit localized to the nucleus, suggesting that accumulation of catalytic subunit in the nucleus requires either enzymatic activity or a nuclear localization signal. Inactivation of the catalytic subunit in vitro by treatment with N-ethylmaleimide did not prevent localization in the nucleus, indicating that enzymatic activity was not a prerequisite for nuclear localization. In an effort to search for a specific signal that might mediate nuclear localization, a complex of the catalytic subunit with a 20-residue inhibitory peptide derived from PKI (PKI(5-24)) was microinjected. In contrast to intact PKI, the peptide was not sufficient to block nuclear accumulation. In the presence of PKI(5-24), the C subunit localized to the nucleus in a fashion analogous to that of dissociated, active C subunit despite evidence of no catalytic activity in situ. Thus, nuclear localization of the C subunit appears to be independent of enzymatic activity but most likely dependent upon a signal. The signal is apparently masked by both the regulatory subunit and PKI but not by the inhibitory peptide.

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Refeeding starved chicks causes a 25- to 50-fold increase in the level of malic enzyme mRNA in liver. To define the regulated steps, we measured transcriptional activity of the malic enzyme gene using the nuclear run-on assay and a variety of DNA probes specific to the malic enzyme gene. Refeeding starved chicks stimulated transcription of the malic enzyme gene in liver by 40- to 50-fold. An increased transcription rate was detectable at 1.5 h, was maximal at 3 h, and remained high at 24 h of refeeding. The level of nuclear precursor RNA for malic enzyme assessed by hybridization with intron-specific probes was high in liver of refed birds, and barely detectable in that of starved birds. These results indicate that nutritional regulation of the level of malic enzyme mRNA is transcriptional. Low levels of malic enzyme mRNA in brain, kidney, and heart correlated well with low rates of transcription of the malic enzyme gene in these tissues. In contrast to liver, neither the rate of transcription nor the steady-state level of malic enzyme mRNA was affected by refeeding starved birds. A series of DNase I-hypersensitive sites were located within 4000 base pairs upstream of the transcription start site of the malic enzyme gene in liver. The DNase I-hypersensitive region extending from the start of transcription to 400 base pairs upstream was much more pronounced in the refed state than in the starved state. This change in DNase I hypersensitivity followed the same time course as increased transcription of the malic enzyme gene. This DNase I-hypersensitive region also was present at low intensity in kidney and heart independently of nutritional state. The three constitutive DNase I-hypersensitive sites further upstream were present in liver but not in kidney or heart.

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Sensitive immunochemical assays were used to measure the mass and rate of synthesis of malic enzyme protein in wild-type and Mod-1n mutant mice fed a high carbohydrate/low fat diet supplemented with thyroid hormone. Malic enzyme activity in the fed, wild-type mice was 100-fold higher than in starved, wild-type mice. Neither activity, mass, nor synthesis of malic enzyme could be detected in fed, mutant mice. However, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase responded to these dietary manipulations with normal or supranormal increases in activities, respectively, in mutant mice. A cDNA clone containing an almost complete copy of the mRNA for malic enzyme from duck liver was used to analyze poly(A+) RNA from C57BL/6J-DBA/2J hybrid mice that had been fasted and refed a high carbohydrate/low fat diet supplemented with thyroid hormone. The 32P-cDNA probe hybridized to two RNAs of 2250 and 2950 nucleotides. The same two RNAs were detected in RNA from starved mice except at much lower concentrations. A similar analysis of RNA from Mod-1n mice fed the high carbohydrate-thyroid diet also revealed two hybridizing RNAs but each was 700-800 nucleotides longer than its counterpart in wild-type mice. The abundance of malic enzyme mRNA in the fed, mutant mice was about the same as that in fed, wild-type mice. The mutant malic enzyme mRNAs also were present in RNA from starved mice but at much lower concentrations. These results suggest that the mutation responsible for the Mod-1n phenotype is in the structural gene for malic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

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