L-type Pyruvate Kinase mRNA Research Articles

Dietary and hormonal regulation of L-type pyruvate kinase gene expression in rat small intestine.

L-type pyruvate kinase is an enzyme of the glycolytic pathway whose activity and mRNA levels fluctuate in the small intestine according to dietary status. Both the enzyme activity and mRNA concentration decline during fasting and increase upon refeeding either a glucose-rich or a fructose-rich diet. Using a single-strand M 13 phage complementary to L-type pyruvate kinase mRNA as probe, we determined the level of the mRNA in the small intestine of normal, adrenalectomized, thyroidectomized, diabetic and glucagon-treated or cAMP-treated animals refed either a glucose-rich or a fructose-rich diet. The specific mRNA is present in the small intestine of normal fasted rats and increases twofold and threefold on refeeding glucose and fructose respectively. However, the hormonal control of the gene expression differs according to the dietary carbohydrate. The L-type pyruvate kinase mRNA increase, induced by glucose feeding, is hormone-dependent and requires the presence of thyroid hormones and insulin. In fructose-fed rats a certain level of mRNA increase occurs regardless of the hormonal status of the animals, but the full induction of the mRNA by fructose requires the presence of glucocorticoids, thyroid hormones and insulin. Thus, the hormonal regulation of L-type pyruvate kinase gene expression in the small intestine is largely similar to that described in normal rat liver but the basal mRNA level and the stimulation of the mRNA increase by fructose are higher in the small intestine.

Effects of chronic renal failure on the regulation of pyruvate kinase

The effects of chronic renal failure on the enzyme activity of pyruvate kinase and the mRNA level of this enzyme were studied in 7 out of 8 nephrectomized rats. The mRNA level was measured by RNA-DNA dot blot hybridization, using cloned pyruvate kinase cDNA as hybridized probe. Neither the activity of M 1-type pyruvate kinase nor the level of this enzyme in rat gastrocnemius muscle was affected by chronic renal failure, whereas l-type pyruvate kinase enzyme activity in uremic rat liver was lower than that in control at both fasted and refed states. The levels of l-type pyruvate kinase mRNA were not different between two groups as the fasted state. Induction of l-type pyruvate kinase mRNA after high carbohydrate diet refeeding was suppressed proportionally to the severity of chronic renal failure, which was expressed by the serum creatine concentrations ( r = −.876, P < .005). These results indicate that the suppression of l-type pyruvate kinase activity in uremia was partly reflected by the decreased accumulation of this enzyme mRNA. There was a significantly negative correlation between l-type pyruvate kinase mRNA levels and plasma glucagon/insulin ratios ( r = −.719, P < .05). Hyperglucagonemia in uremia might play a major role in this suppression.

Early modifications of gene expression induced in liver by azo-dye diet

The expression and regulation of the phosphoenolpyruvate carboxykinase gene were not grossly modified by feeding rats a 3'-methyl-4-(dimethylamino)azobenzene-containing diet despite maximum expression of the L-type pyruvate kinase gene being dramatically reduced as early as the 24th hour of the carcinogenic diet. Inhibition of aldolase B mRNA synthesis occurred more slowly, being maximum at the 3rd day. After stopping administration of the carcinogen, a very rapid, but transient increase of the L-type pyruvate kinase mRNA was observed at the 24th hour, whereas aldolase B mRNA increased only slowly. The amount of aldolase A mRNA fell quickly after termination of carcinogen administration, levels being normal at the 2nd–3rd day. At this time, the histological structure of the liver was indistinguishable from that of animals still receiving the azo-dye diet. It appears, therefore, that in the rat both administration and withdrawal of the azo-dye carcinogen induce rapid modifications of the expression of some genes, before any cellular modification is distinguishable.

Expression of brain-specific ID sequence is not restricted to the brain, and a novel complementary cID sequence is found in L-type pyruvate kinase mRNA (a liver-specific messenger)

Expression of brain-specific ID sequence is not restricted to the brain, and a novel complementary cID sequence is found in L-type pyruvate kinase mRNA (a liver-specific messenger)

Tissue-specific heterogeneity of the 3'-untranslated region of L-type pyruvate kinase mRNAs.

A single L-type pyruvate kinase (PK) gene seems to exist per haploid genome. It is expressed in the liver, kidney and small intestine in the form of three mRNA species of 2, 2.2 and 3.2 X 10(3) bases (kb). All three species are polyadenylated and translatable into the same L-type subunit. Primer extension experiments demonstrate that all three PK mRNAs have the same 5' ends. Nuclease S1 protection experiments with various cDNA and 3' genomic probes indicate that the different mRNA species only differ by the length of their 3' noncoding region. The mechanism responsible for the production of the three transcripts seems to be the use of alternative unusual polyadenylation sites. Run-on assays with specific probes recognizing only the 3.2-kb or all three mRNA species show that the transcription proceeds across the gene with similar rate. This means that the process involved in generation of the three transcripts is a posttranscriptional event, probably due to different sites of endonucleolytic cleavage of primary transcripts extending 3' from the gene region encoding the mature mRNAs. The ratio between the different PK mRNA species is, to a certain extent, tissue-specific and changes with development. The role of an 'identifier sequence' located in the 3' noncoding sequence of the 3.2-kb species in such a tissue-specific use of alternative polyadenylation sites is discussed.

Transcriptional and post-transcriptional regulation of L-type pyruvate kinase gene expression in rat liver.

The effects of starvation, refeeding a diet high in carbohydrate, administration of glucagon and cyclic AMP, thyroidectomy, and adrenalectomy on transcription of the gene for liver L-type pyruvate kinase and on the accumulation of cytoplasmic mRNA for L-type pyruvate kinase were investigated in rat. Transcription of the gene was undetectable in either fasted or protein-fed rats. Refeeding fasted rats a carbohydrate-rich diet stimulated an increase in L-type pyruvate kinase mRNA, preceded by an increase in the gene transcription. Transcription was maximal at 12 h of refeeding, decreasing to 10% of maximum at 72 h. The level of L-type pyruvate kinase mRNA remained constant at 50% of maximum for at least 120 h. Neither thyroidectomy nor adrenalectomy affected gene transcription in fasted rats refed the carbohydrate-rich diet, despite a decrease in mRNA abundance to 40 and 20%, respectively, of controls fed a normal diet. Glucagon or cyclic AMP totally blocked the increase in transcription of the L-type pyruvate kinase gene caused by feeding a carbohydrate-rich diet to previously fasted rats. Nevertheless, the level of L-type pyruvate kinase mRNA remained high for 3 h after glucagon administration. After 3 h, the mRNA decreased rapidly with a half-life less than 1 h. Thus, expression of the gene for L-type pyruvate kinase is regulated at both transcriptional and post-transcriptional levels. The transcription is regulated by two major effectors, one positive, namely carbohydrates, and one negative, namely glucagon (via cyclic AMP). Both agents probably act at the level of the mRNA stability as well. Glucocorticoids and thyroid hormones do not regulate transcription of the gene for L-type pyruvate kinase but do appear to be required for a normal accumulation of the transcripts in the cytoplasm.

Sequences complementary to the brain-specific “identifier” sequences exist in L-type pyruvate kinase mRNA (a liver-specific messenger) and in transcripts especially abundant in muscle.

A sequence complementary to the brain-specific identifier sequence has been found in the 3' untranslated extension of the heavy 3.2-kilobase (kb) long liver L-type pyruvate kinase mRNA while it is absent in the other two 2- and 2.2-kb long pyruvate kinase mRNA species. A 53-base fragment corresponding to this identifier sequence was subcloned in both orientations in the single-stranded bacteriophage M13, both strands being used as probes to detect homologous sequence in different tissues. Both strands are transcribed in various tissues and are detected in heterogeneous high molecular weight RNA species which are especially abundant in the adult muscle. In addition, the probe identical to the identifier sequence recognized a discrete 0.6-kb RNA species in the muscle and the probe complementary to the identifier-sequence recognized the expected two small brain-specific identifier BC-1 and BC-2 RNAs described by Sutcliffe et al. (Sutcliffe, J. G., Milner, R. J., Bloom, F. E., and Lerner, R. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4942-4946; Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M., and Lerner, R. A. (1984) Nature 308, 237-241; Milner, R. J., Bloom, F. E., Lai, C., Lerner, R. A., and Sutcliffe, J. G. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 713-717; Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M., and Reynolds, W. (1984) Science 225, 1308-1314).

Modifications of the expression of liver‐specific and non‐specific messenger RNAs during azo‐dye hepatocarcinogenesis

The expression of specific and non-specific rat liver messenger RNAs has been studied during 3'-methyl-4-(dimethylamino)azobenzene (3'-MeDAB) carcinogenesis, using cDNA probes complementary to mRNAs encoding aldolase A and B, L-type pyruvate kinase, albumin, alpha-fetoprotein, transferrin and an unidentified 2.7 X 10(3)-base mRNA. mRNAs specific for undifferentiated cells, such as those encoding aldolase A and the unidentified 2.7 X 10(3)-base species were re-expressed very early, being easily detectable at the 1st week of 3'-MeDAB treatment. They reached a maximum of expression at the 4th week. Simultaneously the levels of aldolase B and L-type pyruvate kinase mRNAs dramatically decreased as compared to controls, but remained responsive to induction by a high-carbohydrate diet. Albumin and transferrin mRNA levels were only slightly modified in the course of the carcinogenic diet. At the terminal stage of hepatocarcinogenesis, i.e. in malignant hepatoma cells, expression and inducibility of aldolase B and L-type pyruvate kinase mRNAs were similar to those in normal adult rats while mRNAs specific for undifferentiated or foetal stages were also synthesized. The very early changes in gene expression for aldolases A and B, L-type pyruvate kinase and the 2.7 X 10(3)-base mRNA species could indicate that carcinogenic diet modifies gene control mechanisms long before inducing hepatoma.

Rapid regulation of L-type pyruvate kinase mRNA by fructose in diabetic rat liver.

The effect of fructose on the induction of L-type pyruvate kinase mRNA in diabetic rat liver was studied by using a cloned cDNA probe. Fructose feeding resulted in a 5- to 6-fold increase in the L-type enzyme mRNA level after 1 to 3 days. These changes were approximately proportional to the changes in the level of translatable mRNA of this enzyme. A significant increase in total cellular L-type enzyme mRNA level was observed within 2 h after fructose feeding and the level reached a maximum after 8 h. Dietary glycerol also markedly increased the L-type mRNA level. These alterations were essentially due to the changes in the cytosolic mRNA. Northern blot analysis of total cellular RNA revealed that two L-type enzyme mRNA species with molecular sizes of 2.1 and 3.6 kilobases were proportionally increased during the fructose induction. The two mRNA forms were found in immunopurified L-type enzyme mRNA and directed synthesis of the L-type subunit in vitro; they are therefore functional mature forms. In contrast, analysis of nuclear RNA showed five putative precursor RNA species for the enzyme, up to 9.4 kilobases in length, in the liver of fructose-fed rats, while no band of the RNA species was found in the nuclei of control liver. The changes in the number of bands of these RNA species and their intensities after fructose feeding preceded the changes in the level of total cellular L-type enzyme mRNA sequences. These results indicate that dietary fructose causes a rapid increase in the level of L-type pyruvate kinase mRNA sequences by acting at the nuclear level.

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.

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.

Molecular cloning of DNA complementary to rat L-type pyruvate kinase mRNA. Nutritional and hormonal regulation of L-type pyruvate kinase mRNA concentration.

Rat liver L-type pyruvate kinase mRNA was enriched from total polysomes by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to L-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-selected translation, and dot blot hybridization. A partial restriction endonuclease map of cDNA inserts was constructed covering about 1.86 kilobase pairs. The cDNA insert of recombinant plasmid pLPK-14 was used as a hybridization probe to quantitate L-type pyruvate kinase mRNA in rat liver after various treatments. The level of hybridizable L-type enzyme mRNA was markedly increased by a high carbohydrate diet. Diabetes greatly reduced the mRNA level in the liver of rats maintained on a high carbohydrate diet, but insulin administration resulted in restoration of the mRNA level to normal within 24 h. These changes were approximately proportional to the changes in the level of translatable L-type pyruvate kinase mRNA. Thus, we conclude that nutritional and hormonal regulation of synthesis of hepatic L-type pyruvate kinase occurs at the pretranslational level.

Regulation of rat liver L-type pyruvate kinase mRNA by insulin and by fructose.

The effects have been studied of streptozotocin-induced diabetes and subsequent insulin administration or feeding of a high fructose diet on the amount of enzyme protein and mRNA activity of L-type pyruvate kinase in rat liver. Diabetes markedly decreased the L-type enzyme activity in rat liver and insulin treatment resulted in restoration of the enzyme activity to normal. A high fructose diet also increased the enzyme activity in diabetic rats but to a lesser extent. Immunochemical analysis showed that these alterations in the enzyme activity were due to changes in the amount of immunoreactive enzyme protein. The mechanism of the changes was studied further by assaying the level of functional mRNA coding for this enzyme in a nuclease-treated reticulocyte lysate system with total RNA isolated from rat liver. L-type pyruvate kinase mRNA, expressed as a percentage of the total protein synthesized, was greatly decreased in diabetic rats. Insulin administration resulted in recovery of the mRNA activity to the normal level within 24 h. The lag period before accumulation of translatable mRNA of the L-type enzyme was about 4-5 h. The mRNA activity was also increased in diabetic rats fed a high fructose diet. This fructose effect, which was much smaller than the insulin effect, was maximal after feeding fructose diet for one day. These changes were approximately comparable to the changes in enzyme activity. Thus, it is suggested that regulations of rat liver L-type pyruvate kinase by insulin and by fructose are primarily due to changes in the level of translatable mRNA of this enzyme.