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M P Simon

Publications and source records attributed to M P Simon.

30 records · Page 2Linked to original sources
PubMed1984

Cyclic AMP as a transcriptional inhibitor of upper eukaryotic gene transcription.

Recently, glucagon and its second messenger, cyclic AMP, have been shown to stimulate the transcription rate of several upper eukaryotic genes (1-5). We show here that glucagon can also block gene transcription. Both glucagon and cyclic AMP were found to inhibit the transcription of the genes encoding three liver glycolytic enzymes, including L-type pyruvate kinase and aldolase B. Thus, cyclic AMP proves to be not only an activator but also an inhibitor of gene transcription in eukaryotes.

Animals↗
PubMed1984

In vivo hormonal control of L-type pyruvate kinase gene expression. Effects of glucagon, cyclic AMP, insulin, cortisone, and thyroid hormones on the dietary induction of mRNAs in the liver.

Using a cDNA probe complementary to rat L-type pyruvate kinase mRNAs, we studied the respective roles of glucocorticoids, thyroid hormones, glucagon, and insulin in the induction of specific mRNAs in the liver of animals refed either a maltose-rich or a fructose-rich diet. Neither adrenalectomized nor thyroidectomized nor diabetic animals could express L-type pyruvate kinase mRNAs in their liver when refed the carbohydrate-rich diets. When the animals were given the missing hormone, the level of hybridizable mRNAs returned to normal values but administration of the hormone alone failed to induce mRNA synthesis in fasted animals. Both glucagon and cyclic AMP abolished the induction of L-type pyruvate kinase mRNAs in refed animals. Exogenous insulin, whatever the dose, could not reverse the inhibitory action of glucagon. Insulin has usually been regarded as the main regulator of L-type pyruvate kinase gene expression. It appears now that glucagon, beside regulating the enzyme activity by phosphorylation mechanisms, may also modulate L-type pyruvate kinase synthesis at a pre-translational level. Consequently, our results show that three conditions are required for the synthesis of liver L-type pyruvate kinase mRNAs: (i) the presence of dietary carbohydrates, (ii) the cessation of glucagon release, and (iii) the presence of permissive hormones, including insulin.

Adrenalectomy↗
PubMed1984

Dietary control of aldolase B and L-type pyruvate kinase mRNAs in rat. Study of translational activity and hybridization with cloned cDNA probes.

Liver L-type pyruvate kinase and aldolase B mRNAs are the two species whose translational activity increases the most after feeding starved rats a high carbohydrate diet (Simon, M. P., Besmond, C., Cottreau, D., Weber, A., Chaumet-Riffaud, P., Dreyfus, J. C., Sala Trépat, J., Marie, J., and Kahn, A. (1984) J. Biol. Chem., in press). We therefore compared the pattern of this induction in three tissues synthesizing these enzymes, e.g. the liver, small intestine, and kidney. Influence of high lipid and protein diets on liver L-type pyruvate kinase and aldolase B mRNAs was also investigated. In the starved rat livers, L-type pyruvate kinase mRNA was practically undetectable. Carbohydrate diet induced an increase of both mRNA concentrations, with a maximum at the 12-18th h; at this time, mRNA concentration was increased about 4-8 times for aldolase B and 40-100 times for L-type pyruvate kinase, translational activities representing about 1% of the total mRNA activity for both enzymes. After the 24th h of carbohydrate diet, mRNA concentrations decreased slightly, then remained in plateau. In animals refed the high carbohydrate diet, starvation as well as high lipid and protein diets provoked a rapid decrease of both mRNA concentrations and translational activities. In the kidney, aldolase B mRNA synthesis was high in starved rats and was only slightly stimulated by carbohydrates (1.5-2.5 times). L-type pyruvate kinase mRNA concentration was increased 6-15-fold after feeding a high carbohydrate diet. In the small intestine, in contrast, the extent of aldolase B mRNA induction by a carbohydrate diet was similar to that in the liver, while L-type pyruvate kinase mRNA concentration was practically similar in starved and refed rats (about 1:10 of the concentration observed in refed rat liver). These results seem to indicate that the mechanisms responsible for carbohydrate induction of L-type pyruvate kinase and aldolase B are different. In addition, dietary control of each enzyme is also different in the various tissues which synthesize them.

Animals↗
PubMed1983

Molecular cloning of cDNA for rat L-type pyruvate kinase and aldolase B.

Two double-stranded cDNA recombinant pBR322 plasmid libraries were constructed starting from high carbohydrate diet rat liver poly(A)+ mRNA, either fractionated by denaturing sucrose gradient centrifugation for the cloning of L-type pyruvate kinase cDNA, or nonfractionated for aldolase B. Both libraries were screened with single-stranded cDNA probes reverse transcribed from fasted or high carbohydrate diet rat liver mRNAs. mRNAs from fasted animals were also fractionated by sucrose gradient centrifugation and mRNAs from the fed animals were, in addition, further purified by high performance liquid gel filtration chromatography. Those clones hybridizing with the "positive" probe (from animals fed the high carbohydrate diet) and not with the "negative" one (from fasted animals) were preselected and their plasmid DNA was purified and analyzed by positive hybridization-selection. Thirty of 4500 bacteria colonies transformed by recombinant plasmids were preselected by differential screening for pyruvate kinase, and 8 of 864 colonies for aldolase B. Twenty-two recombinant plasmids for pyruvate kinase and two for aldolase B were shown to contain specific cDNA inserts by positive hybridization-selection. Plasmids DNAs of some pyruvate kinase and aldolase B clones (whose inserts ranged from 700 to 1050 bases in length) were labeled by nick translation and used as probes for Northern blot hybridization. The pyruvate kinase cDNA probes recognized mainly a 3400-base RNA species which was detected in high carbohydrate diet rat liver, but not in fasted rat liver and in tissues which do not synthesize L-type pyruvate kinase. In addition, some pyruvate kinase probes hybridized with minor RNA species of about 2000 bases in length, only observed after carbohydrate diet. For aldolase B, the recombinant plasmid DNA hybridized with a single RNA species of 1750 bases. This RNA, detected in kidney, small intestine and liver, was induced by a high carbohydrate diet and increased with liver development. The rat probe cross-hybridized with human aldolase B messenger RNA.

Animals↗
PubMed1983

Genetic and epigenetic control of the pyruvate kinase isozymes in mammals.

In man, patients with hereditary red cell pyruvate kinase (PK) deficiency have their enzyme concomitantly affected in both red cells and liver (PK-L), such that red cell L' and liver L PK subunits appear to be encoded by the same structural gene. Very recently, a mutant M1 PK has been described in muscle from a special mouse strain in which the M2 PK was concomitantly modified. Thus, it has been suggested that the M1 and M2 PK subunits are also encoded by the same structural gene, a gene distinct from that encoding L' and L. In this paper, we show that synthesis of each of the four PK subunits is specified by a different specific mRNA. Two structural genes, but at least four different mRNAs seem therefore to control synthesis of the PK isozymes. Genetic and epigenetic mechanisms responsible for generation of this diversity at the RNA level are discussed. In addition to these mechanisms of PK diversity, some PK subunits can be modified by post-translational events. In red cells, for instance, cell aging is associated with a progressive, partial proteolysis of the C-terminal extremities of L' subunits, ultimately resulting in cleavage of the phosphorylatable site.

Animals↗
PubMed1982

Molecular organization of human L' and L pyruvate kinases.

Extremities, peptide maps and phosphorylatable site localization of human erythrocyte L' and liver L pyruvate kinases (EC 2.7.1.40) were investigated. L' and L subunits seemed to have similar, blocked NH2 termini and differ in their sensitivity to carboxypeptidase A, that is to say in their C-terminal ends. After digestion by Staphylococcus aureus V8 protease, the phosphorylated sites of both L' and L subunits were located on those peptides which were different in L' and L, that is to say on the C-terminal sides. A mild proteolytic attack of the native tetrameric enzymes by trypsin partially degraded the phosphorylatable peptides without removing the phosphoserine residue; in the same conditions, chymotrypsin split off this phosphorylated residue and subtilisin totally degraded the phosphorylated peptides. From these results it appears, therefore, that age-dependent proteolytic degradation of L' subunits in old red cells involves the C-terminal side of the molecules, ultimately resulting in cleavage of the phosphorylated site. Since erythrocyte L' and liver L subunits are encoded by different species of messenger RNAs, our results indicate, in addition, that these messenger RNA species should differ by their 3' coding sequences.

Carboxypeptidases↗
PubMed1980

Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinases. Comparison with human liver L-type enzyme.

Human red cell contain soluble adenosine-3',5'-phosphate-dependent protein kinases, which are able to phosphorylate the L' subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a 'T' allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin-treated L'4 enzyme and accounts for less than 20% of total pyruvate kinase subunits. Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.

Cyclic AMP↗
PubMed1979

Preparation of parietal peritoneum for measurements of in vitro permeability.

An experimental method for in vitro studies of permeability of parietal peritoneal membrane was developed in rats. Sodium and potassium fluxes across the peritoneum lining two different regions (the diaphragm and the anterior abdominal wall) were estimated and electrical resistance of the isolated membranes were determined. A significant reciprocal correlation was observed between the two indices. Quantitative differences between the two types of membranes were found, the diaphragmatic region being more permeable than the other. It is suggested that a monolayer of mesothelial cells covering the membranes is partially responsible for these differences. The parietal peritoneum was found to be less permeable than visceral peritoneum (mesentery) studied under identical conditions.

Abdominal Muscles↗