L-type Pyruvate Kinase mRNA Research Articles

Insulin Induces the Expression of the SHARP-2/Stra13/DEC1 Gene via a Phosphoinositide 3-Kinase Pathway

Transcription of the rat fatty acid synthase (FAS) gene in the rat liver can be regulated by feeding a high carbohydrate diet. A carbohydrate response element (ChoRE) located on the rat FAS gene promoter has been identified. Using multiple copies of the ChoRE as the bait in a yeast one-hybrid system, a rat liver cDNA library was screened, and the cDNA of ChoRE-binding proteins was cloned. A positive clone that encodes a basic helix-loop-helix protein, enhancer of split- and hairy-related protein-2 (SHARP-2), was obtained. Northern blot analysis revealed that the levels of SHARP-2 mRNA increase when a high carbohydrate diet is fed to normal rats or when insulin is administered to diabetic rats. In primary cultured rat hepatocytes, insulin rapidly induced an accumulation of SHARP-2 mRNA even in the absence of glucose. A time course for the increase in SHARP-2 mRNA levels indicated that it followed by those of FAS and L-type pyruvate kinase mRNAs and that the initial time course of SHARP-2 mRNA was similar to changes in the levels of glucokinase mRNA and phosphoenolpyruvate carboxykinase mRNA. Although wortmannin, LY294002, and actinomycin D blocked the increase in SHARP-2 mRNA levels by insulin, rapamycin, staurosporine, PD98059, okadaic acid, and 8-bromocyclic AMP had no effect. In addition, nuclear run-on assay revealed that transcription of the rat SHARP-2 gene was induced by insulin. Thus, we conclude that insulin induces the transcription of the rat SHARP-2 gene via a phosphoinositide 3-kinase pathway.

Stimulation of Glucose-6-phosphatase Gene Expression by Glucose and Fructose-2,6-bisphosphate

Glucose-6-phosphatase, a key enzyme in the homeostatic regulation of blood glucose concentration, catalyzes the terminal step in gluconeogenesis and glycogenolysis. Glucose, the product of the glucose-6-phosphatase reaction, dramatically increases the level of glucose-6-phosphatase mRNA transcripts in primary hepatocytes (20-fold), and the maximum response is obtained at a glucose concentration as low as 11 mM. Glucose specifically increases glucose-6-phosphatase mRNA and L-type pyruvate kinase mRNA. In the rat hepatoma-derived cell line, Fao, glucose increases the glucose-6-phosphatase mRNA only modestly (3-fold). In the presence of high glucose concentrations, overexpression of glucokinase in Fao cells via recombinant adenovirus vectors increases lactate production to the level found in primary hepatocytes and increases glucose-6-phosphatase gene expression by 21-fold. Similar overexpression of hexokinase I in Fao cells with high levels of glucose does not increase lactate production nor does it change the response of glucose-6-phosphatase mRNA to glucose. Glucokinase overexpression in Fao cells blunts the previously reported inhibitory effect of insulin on glucose-6-phosphatase gene expression in these cells. Raising the cellular concentration of fructose-2,6-bisphosphate, a potent effector of the direction of carbon flux through the gluconeogenic and glycolytic pathways, also stimulated glucose-6-phosphatase gene expression in Fao cells. Increasing the fructose-2,6-bisphosphate concentration over a 15-fold range (12 +/- 1 to 187 +/- 17 pmol/plate) via an adenoviral vector overexpression system, led to a 6-fold increase (0.32 +/- 0. 03 to 2.2 +/- 0.33 arbitrary units of mRNA) in glucose-6-phosphatase gene expression with a concomitant increase in glycolysis and a decrease in gluconeogenesis. Also, the effects of fructose-2, 6-bisphosphate concentrations on fructose-1,6-bisphosphatase gene expression were stimulatory, leading to a 5-6-fold increase in mRNA level over a 15-fold range in fructose-2,6-bisphosphate level. Liver pyruvate kinase and phosphoenolpyruvate carboxykinase mRNA were unchanged by the manipulation of fructose-2,6-bisphosphate level.

Modulation of glucose responsiveness of insulinoma beta-cells by graded overexpression of glucokinase.

Insulinoma beta-cells capable of overexpressing glucokinase under the control of a doxycycline-dependent transcriptional transactivator were established from parental INS-1 cells. Glucokinase could be maximally induced to a level more than 20 times the basal level after 36 h of culture with doxycycline. Intermediate levels of induction could be achieved by varying doses of, and time of culture with, the inducer. The rate of glycolysis was measured in cells with 3-, 5-, and 8-fold increment in glucokinase activity above the noninduced level. Proportionate increases in glycolytic flux occurred in cells cultured at low physiological glucose concentration. At high glucose concentration, induction of glucokinase in excess of 2-fold above basal resulted in little additional increase in glycolysis. The consequences of graded increases of glucokinase on two physiological glucose effects were investigated. Increments in glucokinase activity were accompanied by a stepwise shift to the left of the dose-response curve for the inductive effect of glucose on the L-type pyruvate kinase mRNA. Similarly, the insulin secretory response to glucose was shifted leftward in glucokinase-induced cells. The following conclusions are drawn: (i) glucokinase is the major rate-limiting enzyme for glycolysis in these cells; (ii) downstream metabolic steps become limiting at high extracellular glucose concentration with moderate increases in glucokinase over the wild-type level; (iii) within limits, glucokinase activity is a determining factor for two types of glucose responses of the beta-cell, the induction of specific gene expression, and insulin release.

Disordered expression of glycolytic and gluconeogenic liver enzymes of juvenile visceral steatosis mice with systemic carnitine deficiency

A quantitative study of the effect of carnitine deficiency on expression of glycolytic and gluconeogenic enzymes was performed using juvenile visceral steatosis mice which are systemically deficient in carnitine. The amounts of glucokinase and L-type pyruvate kinase mRNA were reduced in homozygotes, compared to heterozygotes and normal controls at 2 and 8 weeks. Liver-type phosphofructokinase, however, did not differ significantly. The abundance of fructose 1,6-bisphosphatase mRNA was unchanged at 2 and 8 weeks. The level of phosphoenolpyruvate carboxykinase mRNA was increased slightly at 2 weeks, but not at 8 weeks. A part of these changes could not be explained by the plasma glucose or insulin level. Carnitine administration restored the mRNA of these enzymes to normal levels. These results suggest that carnitine deficiency affects the expression of these liver enzymes.

Relationship between the concentrations of glycolytic intermediates and expression of the L-type pyruvate kinase gene in cultured hepatocytes.

Previous studies have suggested that some glycolytic intermediates are involved in the regulation of L-type pyruvate kinase gene expression by carbohydrates such as glucose and fructose. To find such intermediates, we examined the relationship between the levels of L-type pyruvate kinase mRNA and glycolytic metabolites in hepatocytes cultured under various conditions. Of the metabolites, the levels of 3-phosphoglycerate and phosphoenol-pyruvate only increased significantly under conditions under which the expression of the L-type pyruvate kinase gene was stimulated. The level of glucose 6-phosphate, which was reported to be involved in dietary stimulation of this gene expression, was not correlated with the mRNA level since marked accumulation of deoxyglucose 6-phosphate occurring on the addition of deoxyglucose, a nonmetabolizable glucose analog, was not accompanied by an increase in the L-type pyruvate kinase mRNA level. In addition, we found that fructose at a low concentration in the presence of glucose failed to increase the mRNA and metabolite levels in contrast to other reports that the promoter activity of the L-type pyruvate kinase gene is stimulated by this treatment. Thus we propose that 3-phosphoglycerate and/or phosphoenolpyruvate are involved in the carbohydrate regulation of L-type pyruvate kinase gene expression.

Evidence from transgenic mice that myc regulates hepatic glycolysis.

The product of the c-myc proto-oncogene (c-Myc) is involved in the control of cell proliferation, differentiation, and apoptosis. It acts as a transcription factor that recognizes the CACGTG motif. This sequence has also been found in the glucose-responsive elements of genes involved in the control of liver glycolysis and lipogenesis. To determine whether c-Myc can regulate hepatic carbohydrate metabolism in vivo, transgenic mice that overexpress c-myc under control of the P-enolpyruvate carboxykinase (PEPCK) gene promoter have been generated. These mice showed a threefold increase in c-Myc protein in liver nuclei. Hepatocytes from transgenic mice were normal and did not acquire the fetal phenotype. However, transgenic mice showed higher levels (threefold) of L-type pyruvate kinase mRNA and enzyme activity than control mice. The increase in pyruvate kinase activity led to a three- to fivefold increase in liver lactate content and a fivefold induction of lactate production by hepatocytes in primary culture. The expression of the 6-phosphofructo-2-kinase gene was also increased in the liver of these transgenic mice. The induction of hepatic glycolysis was related with an increase in the expression (about fourfold) and activity (about threefold) of liver glucokinase, whereas no change was noted in hexokinase-I. This change in glucokinase activity led to an increase in both glucose 6-phosphate and glycogen contents in the liver of transgenic mice. The expression of the liver-specific glucose transporter GLUT2 was also increased in transgenic mice, whereas no change was noted in the mRNA concentration of GLUT1. Furthermore, the changes of liver glucose metabolism led to a marked reduction of blood glucose (25%) and insulin (40%) concentrations in starvation, whereas the fall in both was only 10% in fed mice. Thus, liver glucose metabolism could determine the blood glucose and insulin set points in the transgenic mice. All these results indicated that the increase in c-Myc protein was able to induce liver glucose utilization and accumulation, and suggested that c-Myc transcription factor is involved in the control in vivo of liver carbohydrate metabolism.

The pyruvate kinase gene as a model for studies of glucose-dependent regulation of gene expression in the endocrine pancreatic beta-cell type.

The insulinoma beta-cell line INS-1 expresses the L-type pyruvate kinase gene at high level and responds to a rise in extracellular glucose by strong induction of gene expression. Following the addition of glucose to the culture medium in the 3.5-33 mM concentration range, the cellular level of L-type pyruvate kinase mRNA increases within 2 h and reaches a maximum 15-fold above basal in 8-12 h. By run-on nuclear assay, the relative transcription rate of the pyruvate kinase gene is shown to increase 4-fold at maximal stimulation, suggesting that both transcriptional and post-transcriptional effects contribute to mRNA accumulation. The glucose effect is totally suppressed by the hexokinase inhibitor mannoheptulose, indicating a requirement for glucose phosphorylation. The mRNA induction is not inhibited in glutamine-free culture medium or by azaserine, suggesting that the hexosamine biosynthetic pathway is not involved. Moreover, metabolism along the glycolytic pathway does not appear to be an absolute requisite, since 2-deoxyglucose partly mimics the inductive effect of glucose. The glucose effect on the pyruvate kinase gene is reversibly antagonized by agents increasing intracellular cAMP. In addition, the effect is highly specific to the pyruvate kinase gene. Neither proinsulin I mRNA nor glucokinase mRNA are increased in glucose-stimulated INS-1 cells. Short term transfection with CAT plasmids driven by the pyruvate kinase L promoter reveals specific glucose-inducible reporter activity with the 183-base pair promoter region upstream of the cap site. Within this region, the previously described L4 cis-acting element is crucial for glucose responsiveness, as demonstrated by the fact that a plasmid with a mutation in this element does not elicit glucose-inducible CAT activity. Induction of L-type pyruvate kinase mRNA occurs in the islets of rats subjected to fasting and carbohydrate refeeding. In conclusion, the L-type pyruvate kinase gene provides an interesting model of glucose-regulated gene in the endocrine beta-cell type.

The regulation of gene expression by insulin is differentially impaired in the liver of the genetically obese-hyperglycemic Wistar fatty rat

The regulation by insulin and carbohydrates of the gene expression of three key enzymes involved in glucose metabolism was studied in the liver of the Wistar fatty rat, a model of obese non-insulin-dependent diabetes mellitus. A high glucose or fructose diet, or insulin administration caused a similar magnitude of increase in the level of L-type pyruvate kinase mRNA in the liver of Wistar fatty rats and their lean littermates. However, the induction of glucokinase mRNA and repression of phosphoenolpyruvate carboxykinase mRNA by dietary glucose or insulin were impaired in the fatty rats, whereas fructose caused a similar decrease in phosphoenolpyruvate carboxykinase mRNA in both types of rats. These results indicate that the regulation of gene expression of glucokinase and phosphoenolpyruvate carboxykinase, but not of L-type pyruvate kinase, by insulin is impaired in the liver of the Wistar fatty rat.

Phlorizin treatment of diabetic rats partially reverses the abnormal expression of genes involved in hepatic glucose metabolism

Liver insulin resistance and glucagon-stimulated hepatic glucose production are characteristics of the diabetic state. To determine the potential role of glucose toxicity in these abnormalities, we examined whether phlorizin treatment of streptozotocin-diabetic rats resulted in altered expression of genes involved in key steps of hepatic glucose metabolism. By inhibiting renal tubular glucose reabsorption, phlorizin infusion to diabetic rats induced normoglycaemia, did not significantly alter low circulating insulinaemia, but caused a marked decrease in hyperglucagonaemia. Glucokinase and L-type pyruvate kinase mRNA levels were reduced respectively by 90% and 70% in fed diabetic rats, in close correlation with changes in enzyme activities. Eighteen days of phlorizin infusion partially restored glucokinase mRNA and activity (40% of control levels), but had no effect on L-type pyruvate kinase mRNA and activity. In contrast to the glycolytic enzymes, mRNA and activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase were increased (10- and 2.2-fold, respectively) in fed diabetic rats. Phlorizin administration decreased phosphoenolpyruvate carboxykinase mRNA to values not different from those in control rats, while phosphoenolpyruvate carboxykinase activity remained 50% higher than that in control rats. The 50% rise in liver glucose transporter (GLUT 2) mRNA and protein, produced by diabetes, was also corrected by phlorizin treatment. In conclusion, we propose that phlorizin treatment of diabetic rats may induce a partial shift of the predominating gluconeogenesis, associated with hepatic glucose overproduction, into glycolysis, by correction of impaired pre-translational regulatory mechanisms. This could be essentially mediated through improved pancreatic alpha-cell function and subsequent lowering of hyperglucagonaemia. These observations suggest that glucagon-stimulated hepatic glucose production may result, in part, from glucose toxicity.

Vanadate treatment of diabetic rats reverses the impaired expression of genes involved in hepatic glucose metabolism: Effects on glycolytic and gluconeogenic enzymes, and on glucose transporter GLUT2

The trace element vanadium is a potent insulinomimetic agent in vitro. Oral administration of vanadate to rats made diabetic by streptozotocin (45 mg/kg i.v.) caused a 65% fall in plasma glucose levels without modifying low insulinemia. We studied whether the hypoglycemic effect of vanadate was associated with altered expression of genes involved in key steps of hepatic glucose metabolism. Glucokinase (GK) and L-type pyruvate kinase (L-PK) mRNA levels were decreased respectively by 90% and 70% in fed diabetic rats, in close correlation with changes in enzyme activities. Eighteen days of vanadate treatment partially restored GK mRNA and activity (40% of control levels), and totally restored L-PK parameters. In contrast to the glycolytic enzymes, mRNA levels and activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK) were increased (15- and 2-fold, respectively) in fed diabetic rats. Vanadate treatment normalized both PEPCK mRNA and activity in diabetic rat liver. The 2-fold increase in liver glucose transporter (GLUT2) mRNA and protein, produced by diabetes, was also corrected by this treatment. In conclusion, oral vanadate given to diabetic rats induces a shift of the predominating gluconeogenic flux, with subsequent high hepatic glucose production, into a glycolytic flux by pretranslational regulatory mechanisms.

Structural analysis of human pyruvate kinase L-gene and identification of the promoter activity in erythroid cells

The human pyruvate kinase (PK) L-gene is organized in 12 exons over 9.5 kilobases, and the first and second exons are specifically transcribed to the R- and L-type PK mRNA. The 5′-flanking region upstream the first exon has two CAC boxes and four GATA motifs within 250 by from the translational initiation codon. Comparison with the rat L-gene revealed four well-conserved elements in the region. The transient transfection demonstrated that the 270-bp upstream region was a powerful promoter in K562 cells, whereas deletion of the distal 150-bp sequences, which included three GATA motifs, resulted in drastic reduction of the activity. When the hypersensitive site 2 of the human β-globin gene locus was joined to the promoter, the activity of the proximal 120-bp region was enhanced. We concluded that the proximal 120-bp region had a basal promoter activity and that the distal 150-bp region acted as a enhancer in erythroid cells.

Molecular mechanism of induction of key enzymes related to lipogenesis.

Key enzymes related to lipogenesis in the liver are induced by a high glucose diet or insulin and suppressed by starvation, diabetes, or glucagon. Most of these enzymes are also induced by dietary fructose, even in diabetic liver. This regulation occurs at the posttranscriptional level as well as at the transcriptional level. We studied extensively the molecular mechanism of induction of L-type pyruvate kinase (LPK). The transcription of the LPK gene in the liver was stimulated by insulin and inhibited by glucagon. This insulin action required ongoing protein synthesis and metabolism of glucose and was enhanced by glucocorticoid. On the other hand, the mechanism of induction of the LPK by dietary fructose depended on plasma insulin levels. Dietary fructose stimulated transcription of the LPK gene in normal rats, whereas it acted mainly at the posttranscriptional level in diabetic rats. These fructose effects were attributable to a common metabolite of fructose and glycerol. The induction of LPK mRNA by dietary glucose was impaired in the liver of Wistar fatty rats, a model of obese non-insulin-dependent diabetes mellitus, but fructose-induced accumulation of the mRNA was not. Studies on transgenic mice indicated that the 5'-flanking region up to -3 kb of the LPK gene contained all cis-acting elements necessary for tissue-specific expression of LPK and its stimulation by diets and insulin. Further analysis using a transient expression assay revealed the presence of three cis-acting elements necessary for expression of LPK in hepatocytes in the region up to -170 kb. However, these elements alone were not sufficient for dietary and hormonal regulation of this enzyme when analyzed in transgenic mice.

Oral administration of vanadate to diabetic rats restores liver 6-phosphofructo-2-kinase content and mRNA.

Vanadate and insulin were administered to diabetic (streptozotocin) rats to compare their effects on the activity and mRNA content of 6-phosphofructo-2-kinase and L-type pyruvate kinase in the liver. The activity of 6-phosphofructo-2-kinase in livers of diabetic rats was about 40% of that found in normal rats. A similar decrease was found for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase content, measured by immunoprecipitation, and for mRNA, measured by hybridization of Northern blots. Administration of vanadate to the diabetic rats led to a progressive recovery of 6-phosphofructo-2-kinase activity, and 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase content and mRNA. This recovery, which was complete after 15 days of oral treatment, was also obtained after 60 h of insulin administration. L-type pyruvate kinase activity and mRNA were also decreased by about 70% in livers of diabetic rats. Both parameters normalized after 15 days of vanadate treatment, whereas insulin administration (60 h) raised L-pyruvate kinase mRNA three-fold above control values. Oral treatment for 15 days with vanadate can thus mimic the effect of insulin on both pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase in livers of diabetic rats.

Alteration in L-type pyruvate kinase gene expression is not associated with the LF-B1 mRNA level

The relation of expression of the LF-B1 gene with the L-type pyruvate kinase (L-PK) mRNA level in rat liver and hepatoma cells was investigated. The L-PK mRNA level in rat liver changed after partial hepatectomy, during development and on intake of a high carbohydrate diet, while the level of LF-B1 mRNA remained unchanged or altered reciprocally. Dedifferentiated AH-130 cells, which did not express L-PK mRNA, expressed LF-B1 mRNA. These results suggest that transcription of the pyruvate kinase L gene is not simply regulated by the level of LF-B1 mRNA.

Vanadate Induction of L-type Pyruvate Kinase mRNA in Adult Rat Hepatocytes in Primary Culture

In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.

Vanadate induction of L-type pyruvate kinase mRNA in adult rat hepatocytes in primary culture

Vanadate induction of L-type pyruvate kinase mRNA in adult rat hepatocytes in primary culture

Glucose-dependent and -independent effect of insulin on gene expression

Insulin action on gene expression can be glucose-dependent or -independent. Accumulation of aldolase B mRNA and of an unidentified 5.4-kilobase mRNA as well as accumulation of L-type pyruvate kinase mRNAs (Decaux, J.F., Antoine, B., and Kahn, A. (1989) J. Biol. Chem. 264, 11584-11590) in cultured hepatocytes isolated from fasted rats require the presence of both glucose and insulin, these agents not being effective individually. In contrast, maintaining the amount of albumin and transferrin mRNAs in these hepatocytes requires the presence of insulin alone, glucose having no effect by itself. Transcription of the albumin gene, investigated by run-on assay, is active in the presence of insulin alone, with or without glucose, whereas transcription of the aldolase B gene is stimulated by glucose and insulin together, but not by insulin or glucose alone. In addition, the stability of the albumin and aldolase B mRNAs in cultured hepatocytes is lowered in the absence of glucose and insulin together as compared to the stability in the presence of one or both agents. These results confirm that transduction of the insulin signal occurs via distinct pathways; one of these pathways could involve a secondary insulin-dependent modification of metabolite concentration, whereas other pathways could be more directly related to the activity(ies) of the occupied insulin receptor.

Regulation of the Gene Expression of Glucokinase and L-Type Pyruvate Kinase in Primary Cultures of Rat Hepatocytes by Hormones and Carbohydrates1

The regulation of the gene expression of two important glycolytic enzymes, glucokinase and L-type pyruvate kinase, by hormones and carbohydrates was studied, in primary cultures of adult rat hepatocytes. Insulin caused time- and dose-dependent increases in the amounts of the mRNAs of the two enzymes in hepatocytes, although glucokinase responded to this hormone faster than L-type pyruvate kinase. The induction of glucokinase mRNA by insulin did not require the presence of glucose itself, but that of the L-type isozyme was dependent on the glucose concentration. For this effect, fructose and glycerol could partially substitute for glucose, but pyruvate and 2-deoxyglucose, a nonmetabolizable glucose analog, could not. The time course of insulin induction in the presence of fructose, but not of glycerol, was similar to that in the presence of glucose. In the presence of glycerol, the mRNA increased in a diphasic manner: the first increase, which probably reflected the effects of fructose and glycerol in normal liver, reached a maximum after 3 h, whereas the second increase corresponded to the increase in the presence of glucose. These results suggested that some metabolite of glucose was required for the insulin-induced increase in L-type pyruvate kinase mRNA. Cycloheximide inhibited the effects of insulin on the two mRNAs, suggesting that ongoing protein synthesis is required in both cases. The addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, also inhibited the effects of insulin. However, phorbol 12-myristate 13-acetate alone did not induce the two mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-specific expression of rat pyruvate kinase L/chloramphenicol acetyltransferase fusion gene in transgenic mice and its regulation by diet and insulin

We produced transgenic mice carrying about 3 kb of the 5′-flanking sequence of the rat pyruvate kinase L gene linked to the chloramphenicol acetyltransferase (CAT) structural gene. Expression of the transgene was observed only in tissues in which the endogenous L-type pyruvate kinase is expressed. Dietary glucose or insulin induced similar increases in the levels of CAT and L-type pyruvate kinase mRNAs in the liver. However, the fructose-induced level of CAT mRNA was about 3- and 6- fold lower than those of endogenous L-type pyruvate kinase mRNA in the liver and kidney, respectively, confirming our previous finding that stabilization of the transcripts of the pyruvate kinase L gene is an important regulatory step in fructose induction, especially in the kidney. Thus we conclude that all the cis-acting elements responsible for tissue-specific expression of the L-type pyruvate kinase and its stimulation by dietary components and insulin are localized in the sequence from about nucleotide −3000 to +37 in the pyruvate kinase L gene.

Regulation of the expression of the L-type pyruvate kinase gene in adult rat hepatocytes in primary culture

Hepatocytes isolated from adult fasted rats and cultured in the presence of thyroid hormones, glucocorticoids, and in a serum-free medium conserve the essentials of their differentiated function and hormonal sensitivity for at least 1 week. In these cells, the gene for L-type pyruvate kinase is expressed only when glucose and insulin are present together, each of them being inactive by itself. Inhibition of the expression of the L-type pyruvate kinase gene which occurs when glucose and/or insulin are removed from the culture medium is not associated with accumulation of the phosphoenolpyruvate carboxykinase mRNA, which argues against the involvement of intracellular cyclic AMP in this phenomenon. Rather, a transcriptional activator, derived from carbohydrate metabolism and accumulating in the presence of insulin, seems to be needed to support the expression of the L-type pyruvate kinase gene. Glucagon, in vitro as in vivo, inhibits production of the L-type pyruvate kinase mRNAs. In addition to their roles on the production of these mRNAs, glucose and insulin on the one hand and glucagon on the other have profound effects on the stability of the L-type pyruvate kinase messengers: the half-life of the mRNA whose production has been blocked by actinomycin D is 1 h in the presence of glucagon and 24 h in the presence of glucose and insulin. Glucagon and glucose/insulin partially antagonize each other's effect on mRNA stability.