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Nutrient gene regulation is an important adaptation allowingsurvival on intermittent food supplies. This adaptative processexists in all species from yeast to mammals. Glucose, the mostabundant monosaccharide in nature, provides a very good exampleof how organisms have developed regulatory mechanisms to copewith a fluctuating level of nutrient supply. In yeast, glucose facil-itates its own use by inducing expression of genes involved in itsmetabolism while repressing that of those involved in the utiliza-tion of alternative carbon sources (for review, see Ref. 1). Themechanisms by which glucose affects gene expression in yeast arenow relatively well understood.In mammals the response to dietary glucose is more complexbecause it combines effects related to glucose metabolism itself andeffects secondary to glucose-dependent hormonal modifications,mainly pancreatic stimulation of insulin secretion and inhibition ofglucagon secretion. In the pancreatic bcells, glucose is the primaryphysiological stimulus for the regulation of insulin synthesis andsecretion. In the liver, glucose, in the presence of insulin, inducesexpression of genes encoding glucose transporters and glycolyticand lipogenic enzymes

Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP. The DNA regulatory sequence responsible for mediating glucose and cyclic AMP responses, called glucose response element (GlRE), consists of two degenerated E boxes spaced by 5 base pairs and is able to bind basic helix-loop-helix/leucine zipper proteins, in particular the upstream stimulatory factors (USFs). From ex vivo and in vivo experiments, it appears that USFs are required for correct response of the L-PK gene to glucose, but their expression and binding activity are not known to be regulated by glucose. A genetic screen in yeast has allowed us to identify a novel transcriptional factor binding to the GlRE, i.e. the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Binding of COUP-TFII to the GlRE was confirmed by electrophoretic mobility shift assays, and COUP-TFII-containing complexes were detectable in liver nuclear extracts. Neither abundance nor binding activity of COUP-TFII appeared to be significantly regulated by diets. In footprinting experiments, two COUP-TFII-binding sites overlapping the E boxes were detected. Overexpression of COUP-TFII abrogated the USF-dependent transactivation of an artificial GlRE-dependent promoter in COS cells and the glucose responsiveness of the L-PK promoter in hepatocytes in primary culture. In addition, a mutated GlRE with increased affinity for USF and very low affinity for COUP-TFII conferred a dramatically decreased glucose responsiveness on the L-PK promoter in hepatocytes in primary culture by increasing activity of the reporter gene in low glucose condition. We propose that COUP-TFII could be a negative regulatory component of the glucose sensor complex assembled on the GlRE of the L-PK gene and most likely of other glucose-responsive genes as well.

In the liver, transcription of several genes encoding lipogenic and glycolytic enzymes, in particular the gene for fatty acid synthase (FAS), is known to be stimulated by dietary carbohydrates. The molecular dissection of the FAS promoter pointed out the critical role of an E box motif, located at position -65 with respect to the start site of transcription, in mediating the glucose- and insulin-dependent regulation of the gene. Upstream stimulatory factors (USF1 and USF2) and sterol response element binding protein 1 (SREBP1) were shown to be able to interact in vitro with this E box. However, to date, the relative contributions of USFs and SREBP1 ex vivo remain controversial. To gain insight into the specific roles of these factors in vivo, we have analyzed the glucose responsiveness of hepatic FAS gene expression in USF1 and USF2 knock-out mice. In both types of mouse lines, defective in either USF1 or USF2, induction of the FAS gene by refeeding a carbohydrate-rich diet was severely delayed, whereas expression of SREBP1 was almost normal and insulin response unchanged. Therefore, USF transactivators, and especially USF1/USF2 heterodimers, seem to be essential to sustain the dietary induction of the FAS gene in the liver.

The calbindin-D9K gene encodes a vitamin D-induced calcium-binding protein that is expressed as a marker of small intestine differentiation. We have shown that 4580 base pairs of its 5' DNA regulatory region can target reporter transgene expression in the intestine and cause this transgene to respond like the endogenous gene to vitamin D active metabolite and that the homeoprotein Cdx2 is bound to the TATA box in the intestine. We now show that the 4580 base pairs construct confers a differentiated pattern of reporter transgene expression in the intestine and that cooperation between the proximal promoter and a distal element located in an opened chromatin structure is responsible for the intestinal expression and vitamin D responsiveness of the transgene. Gel shift and footprinting assays using duodenal nuclear extracts indicate that this distal element contains a Cdx2-binding site. Finally, a mutation in this distal Cdx2-binding site dramatically decreases intestinal expression in transgenic mice. This report, using an in vivo approach, demonstrates the crucial role of Cdx2 for the transcription of an intestinal gene.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a 115-base pair (bp) promoter fragment was able to ensure the brain-specific expression of the chloramphenicol acetyltransferase (CAT) reporter gene in transgenic mice, but only at a low level (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). Here we show that in vivo activation of this promoter at a high level requires cooperation between an upstream 0.6-kilobase pair (kb) fragment and far upstream sequences. In the 0.6-kb region, a 28-bp DNA element is shown to include overlapping in vitro binding sites for POU domain regulatory proteins and for the Winged Helix hepatocyte nuclear factor-3beta factor. An hepatocyte nuclear factor-3beta-binding site previously described in the short proximal promoter fragment is also shown to interact in vitro with POU proteins, although with a lower affinity than the 28-bp motif. Additional binding sites for POU factors were detected in the upstream 0.6-kb sequences. Progressive deletion in this region resulted in decreased expression levels of the transgenes in mice, suggesting synergistic interactions between these multiple POU-binding sites. We propose that DNA elements characterized by a dual binding specificity for both POU domain and Winged Helix transcription factors could play an essential role in the brain-specific expression of the aldolase C gene and other neuronal genes.

The aldolase B gene is transcribed at a high level in the liver, kidney, and small intestine. This high level of gene expression results from cooperation between a weak but liver-specific promoter and an intronic activator. A deletional study of this activator present in the first intron allowed us to ascribe the maximal enhancer function to a 400-base pair (bp) fragment (+1916 to + 2329). This enhancer is highly liver-specific and enhances the activity of heterologous minimal promoters in a position and distance-independent fashion in transiently transfected Hep G2 hepatoma cells. The aldolase B enhancer is composed of two domains, a 200-bp module (Ba) inactive by itself but which synergizes with another 200-bp module (Bb) that alone retains 25% of the total enhancer activity. The Bb sequence is 76% homologous between human and rat genes and contains several binding sites for liver-enriched nuclear factors. By electrophoretic mobility shift assays, we demonstrated that elements 5 and 7 bind hepatic nuclear factor 1 (HNF1), whereas element 2 binds hepatic nuclear factor 4 (HNF4). A functional analysis of the enhancer whose elements have been mutated demonstrated that mutation of any of the HNF1 sites totally suppressed enhancer activity, whereas mutation of the HNF4-binding site reduced it by 80%.

Elevated glucose concentrations stimulate L-pyruvate kinase (L-PK) gene transcription in liver and islet beta-cells. A glucose response element termed the L4 box (two noncanonical E-boxes located -165 and -154 base pairs upstream of the transcriptional start point) has previously been defined within the proximal promoter region of the gene. However, the identity of the transacting factor(s) which binds to this site remains unclear. We have used photon counting digital imaging of firefly luciferase activity to monitor promoter activity continuously in single living islet beta and derived INS-1 cells, and to analyze the molecular basis of the regulation by glucose. L-PK promoter activity, normalized to cytomegalovirus promoter activity using the distinct Renilla reniformis luciferase, was >/=6-fold higher in cells cultured at 16 mM glucose or above compared with cells cultured at 3 mM glucose. Microinjection of antibodies against the ubiquitous transcription factor USF2 inhibited L-PK promoter activity in beta- and INS-1 cells incubated at 30 mM glucose by 71-87%. Anti-USF2 antibodies had a much smaller effect on promoter activity in INS-1 cells cultured at 3 mM glucose, and on the activity of a modified promoter construct lacking an L4 box. These data support the view that glucose enhances L-PK gene transcription in beta-cells by modifying the transactivational capacity of USF2 bound to the upstream L4 box.

Twenty-six different hepatoma cell lines established from cancer-prone transgenic mice exhibited a close correlation between expression of the GLUT 2 glucose transporter and activation of the L-type pyruvate kinase (L-PK) gene by glucose, as judged by Northern blot analyses and transient transfection assays. The L-PK gene and a transfected L-PK construct were silent in GLUT 2(+) cells and active in GLUT 2(−) cells cultured in glucose-free medium. Transfection of GLUT 2(−) cells with a GLUT 2 expression vector restored the inducibility of the L-PK promoter by glucose, mainly by suppressing the glucose-independent activity of this promoter. Culture of GLUT 2(−) cells, in which the L-PK gene is constitutively expressed, in a culture medium using fructose as fuel selected GLUT 2(+) clones in which the L-PK gene responded to glucose. The expression of the L-PK gene in GLUT 2(−) cells cultured in the absence of glucose was correlated with a high intracellular glucose 6-phosphate (Glu-6-P) concentration while under similar culture conditions Glu-6-P concentration was very low in GLUT 2(+) cells. Consequently, a role of GLUT 2 in the glucose responsiveness of glucose-sensitive genes in cultured hepatoma cells could be to allow for Glu-6-P depletion under gluconeogenic culture conditions. In the absence of GLUT 2, glucose endogeneously produced might be unable to be exported from the cells and would be phosphorylated again to Glu-6-P by constitutively expressed hexokinase isoforms, continuously generating the glycolytic intermediates active on the L-PK gene transcription.

The rat Calbindin-D9K (CaBP9K) gene is mainly expressed in intestine, uterus, and lung and is regulated in a complex tissue-specific manner. To analyze the role of potential regulatory elements, previously defined by DNaseI hypersensivity, we made transgenic mice containing truncated rat CaBP9K fusion gene with simian virus 40 large T antigen and the chloramphenicol acetyltransferase as reporter genes. The transgenes contained CaBP9K promoter fragments with 5' end points at -4400, -1011, and -117 base pairs (bp), whereas the 3' end points was at +365 bp. Northern blot analysis of T antigen expression and chloramphenicol acetyltransferase enzyme-linked immunosorbent assay indicated that a positive element, probably the distal intestine-specific DNaseI HS, necessary to target the expression of the transgene in the intestine, is present between -4400 and -1011 bp. The cephalo-caudal gradient of expression of the transgene along the small intestine was similar to those of the endogenous gene, but an ectopic expression of the transgene was observed in the colon. The -1011 transgene was expressed in epithelial alveolar cells of the lung, in renal proximal tubule cells, and in uterine myometrium, as judged from immunocytochemical, histological, and Northern blot analyses. The shortest, -117 construct was only expressed in uterine myometrium, and it was under a strict estrogen dependence like the endogenous gene. Finally, responsiveness to vitamin D in the duodenum was observed with the largest, -4400 construct. Thus, different tissues utilize distinct cis-acting elements to direct and regulate the expression of the rat CaBP9K gene.

The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was an efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter.

International audience; The aldolase C gene product is a glycolytic isoenzyme specifically detected in brain. We have previously defined a short 115-base pair promoter fragment able to confer on a reporter chloramphenicol acetyltransferase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A' and B boxes) were detected by in vitro DNase1 footprinting experiments with brain, fibroblast, or liver nuclear extracts. Both A/A' and B boxes, sharing structural homology, are able to interact with Sp1, Krox20/Krox24 factors and with other proteins (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). In this paper, we describe a new ubiquitous factor termed Ub able to bind the A/A' box. We also delimit a third element (box C) binding a hepatocyte-enriched protein displaced by a hepatocyte nuclear factor 3-specific oligonucleotide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mice carrying different mutant promoters cloned in front of the CAT gene. A promoter containing only box C was totally inactive, suggesting an essential role of the region containing A/A' and B boxes. However, mutations or deletions of either the A/A' or the B box have no significant effect on the CAT gene expression. We therefore hypothesize that the A/A' and B sites may be functionally redundant. Indeed, constructs harboring only one of these two boxes (A/A' or B) linked to the C box displayed a brain-specific CAT activity similar to that obtained with the wild-type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A' and B boxes, was totally inactive, suggesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.

Surfactant protein A (SP-A) is the most abundant protein associated with phospholipids in pulmonary surfactant. There are several lines of evidence that pulmonary and gastrointestinal epithelium produce closely related surface-active materials, although the presence of SP-A in gastrointestinal tract has so far not been reported. Indirect immunofluorescence experiments using different antibodies raised against rat pulmonary SP-A showed that some jejunal and colonic but not gastric epithelial cells positively stained for SP-A. Analysis of the proteins in cell lysates from rat small intestine and colon studied by Western blot revealed several immuno-reactive bands, including the characteristic triplet of 26-, 32-, and 38-kDa monomeric proteins, less strongly labeled than in lung cells, and higher molecular mass forms of 66 and 120 kDa also present in lung cells. The 66- and 120-kDa bands displayed the expected isoelectric pH of SP-A after two-dimensional electrophoresis. Alkylation induced conversion of the 120-kDa form (almost completely) and the 66-kDa form (partly) into the 26-38-kDa monomeric species. The presence of SP-A mRNA in rat stomach, small intestine, and colon was then searched for by conventional cDNA/reverse transcriptase-polymerase chain reaction. Products of appropriate size (372 base pairs) identical to that of pulmonary tissue were amplified in small intestine and colon but not in stomach or in other tissues used as controls. Cloning and sequencing of rat colon SP-A cDNA revealed the same sequence as the one reported for rat lung SP-A. Furthermore, analysis of the transcriptional initiation site of SP-A gene in colon by anchored-polymerase chain reaction showed that transcription was initiated at the same site in both colon and lung. These data, which demonstrate that small intestine and colon express SP-A constitutively and that this protein is present in some epithelial cells, extend the concept of intestinal surfactant and underline its close relationships to pulmonary surfactant.

L-type pyruvate kinase (L-PK) gene transcription is induced by glucose through its glucose response element (GlRE) composed of two degenerated E boxes able to bind in vitro ubiquitous upstream stimulator factor (USF) proteins. Here we demonstrate in vivo, by transient transfections in hepatoma cells, that (i) native USF proteins synthesized from expression vectors can act as transactivators of the L-PK promoter via the GlRE, stimulating transcription without glucose and, therefore, decreasing the glucose responsiveness of the promoter; (ii) expression of the truncated USF proteins, able to bind the GlRE but devoid of the NH2-terminal activation domain, represses the activation of the L-PK promoter by glucose; and (iii) a similar repression of the glucose effect is observed upon expression of mutant USF proteins devoid of the basic DNA binding domain, able to dimerize with endogenous USF but not to bind the GlRE. We conclude that USF proteins are components of the transcriptional glucose response complex assembled on the L-PK gene promoter.

The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway mainly expressed in the liver. Rat liver contains a regulatory protein that inhibits glucokinase (GK) activity. The effect of this protein is greatly reinforced by the fructose 6-phosphate and antagonized by the fructose 1-phosphate (Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184). In hepatocytes, fructose in low concentrations is phosphorylated into fructose 1-phosphate, and therefore is able to active GK in the absence of insulin via the regulatory protein in the liver. In primary culture of rat hepatocytes, 0.2 mM fructose in the presence of 20 or 40 mM glucose stimulated the activity of the L-PK gene promoter fused with the chloramphenicol acetyltransferase reporter gene, regardless of the addition of insulin, through the glucose/insulin response element. A constitutive GK expression vector co-transfected with the L-PK/chloramphenicol acetyltransferase construct is also able to confer an insulin-independent glucose responsiveness in hepatocytes. Thus, the insulin effect on glucose-dependent activation of the L-PK promoter is, under these experimental conditions, to permit glucose phosphorylation through the stimulation of the GK synthesis. In the presence of glucose, the L-PK promoter can also be activated by a post-translational GK activation, mediated by a low concentration of fructose acting via the regulatory protein of glucokinase.

The L-pyruvate kinase (L-PK) gene is slightly active in normal and tumoral endocrine pancreatic tissues while, in vivo, this gene is not transcribed in the exocrine pancreas. Nevertheless, the L-PK gene is re-expressed at a very low level in cultured 266.6 cells derived from an exocrine pancreas carcinoma. The L-PK gene is early activated in endodermal tissues, e.g. yolk sac and primitive intestine; it remains transcribed in fetal pancreas. In adult, L-PK gene expression is restricted to some endocrine cells. Hepatocyte nuclear factor (HNF) 1 and HNF4 are the main tissue-restricted transcription factors involved in tissue-specific expression of the L-PK gene. HNF1 concentration is similar in liver and all pancreatic cells. HNF4 concentration is high in liver, much lower in islets of Langerhans, endocrine pancreatic tumors, and cultured insulinoma cells, and is scarcely detectable in adult exocrine pancreas. This distribution of HNF4 parallels the expression of the L-PK gene. In vivo footprinting experiments show that the HNF1 binding site is similarly occupied in both adult liver and adult pancreas, in which this gene is practically inactive. In this latter tissue, however, the HNF4 binding site is differently occupied with respect to the liver. Since the chromatin structure remains open around the L-PK promoter in pancreas, the L-PK gene can probably be re-expressed under certain circumstances, for instance in cancerous pancreatic cells.

To identify the DNA sequences that cis-regulate the expression of the rat liver pyruvate kinase (L-PK) genes, a series of constructs in which the chloramphenicol acetyltransferase reporter genes is driven by various deleted fragments of the 3200 base pairs (bp) upstream of the L-PK gene cap site have been assayed for transient expression after introduction into hepatoma HepG2 cells, rat hepatocytes in primary culture, fibroblast LTK- cells, myogenic C2C12 cells, and CHO cells. Four distinct regulatory domains have been characterized. A proximal promoter region containing a binding site for the hepatocyte nuclear factor 1 (HNF1) which is sufficient to confer liver specificity, even in the presence of a ubiquitous enhancer. A distal promoter region (-96 to -283 bp) containing binding sites for the liver-specific factor A1 (LFA1), the ubiquitous nuclear factor 1 (NF1), the major late transcriptional factor (MLTF), and so far unidentified proteins binding to the L5-PK region which is essential to maximally activate expression of the construct in HepG2 cells. An extinguisher region, located between positions -2082 and -1170 bp, which decreases efficiency of the L-PK promoter in HepG2 cells, but not in hepatocytes in primary culture. Finally, a far upstream region (-2900 to -2500 bp) which seems to correspond to a liver-specific DNase I hypersensitive site and which behaves in HepG2 cells as an activating sequence efficient in the absence of the extinguisher.

The gene for the L-type pyruvate kinase possesses two promoters which are located 500 base pairs apart. The L promoter is specific to liver and regulated by hormones and diet; the L' promoter is specific to erythroid cells. We produced two series of transgenic mice carrying either the entire rat L-pyruvate kinase gene or a minigene devoid of exons two to nine, with 2.7 kilobases of flanking sequences 5' to the cap site of the L' promoter and 1.4 kilobases 3' to the downstream polyadenylation site. In both series the patterns of expression from the two promoters were similar to those of the endogenous rat gene. The rat L promoter was expressed strongly in liver and weakly in kidney and gut of adult transgenic mice. Moreover, it was regulated like the endogenous rat L-pyruvate kinase gene upon hormonal and nutritional adaptation: the level of L-pyruvate kinase mRNA was decreased dramatically by 24 h of starvation, while refeeding a carbohydrate-rich diet strongly stimulated expression of the transgenes. This stimulation was prevented by glucagon. Use of alternative polyadenylation sites in the last exon of the rat L-type pyruvate kinase gene was similar for both types of transgenes and similar to that in rat and not control mice, suggesting that the transgenes contain the sequences that control the choice of polyadenylation site. Transcription of the minigene was higher than that of the entire transgene, probably due to the high copy number of the minigene. At the protein level, rat L subunits encoded by the entire transgene were more abundant than mouse subunits in the liver of adult transgenic mice. In contrast, expression of the rat L' promoter in fetal liver was only 5% of that in fetal rat liver, and we were unable to detect rat L' subunits of pyruvate kinase enzyme in the red blood cells from transgenic mice. Our results suggest that the integrated DNA contains all elements necessary for tissue specificity (L' and L) as well as hormonal and nutritional control (L) of expression of the rat transgene. Nevertheless, a L'-specific activating element may be missing.

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 Trepat, 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.

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).

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.

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.