L-type Pyruvate Kinase Gene Expression Research Articles

Signaling cross-talk between hypoxia and glucose via hypoxia-inducible factor 1 and glucose response elements

The substrates oxygen and glucose are important for the appropriate regulation of metabolism, angiogenesis, tumorigenesis and embryonic development. The knowledge about an interaction between these two signals is limited. We demonstrated that the regulation of glucagon receptor, insulin receptor and L-type pyruvate kinase (L-PK) gene expression in liver is dependent upon a cross-talk between oxygen and glucose. The periportal to perivenous drop in O2 tension was proposed to be an endocrine key regulator for the zonated gene expression in liver. In primary rat hepatocyte cultures, the expression of the glucagon receptor and the L-PK mRNA was maximally induced by glucose under arterial pO2 whereas the insulin receptor was maximally induced under perivenous pO2. It was demonstrated for the L-PK gene that the modulation by O2 of the glucose-dependent induction occured at the glucose-responsive element (GlcPKRE) in the L-PK gene promoter. The reduction of the glucose-dependent induction of the L-PK gene expression under venous pO2 appeared to be mediated via an interference between hypoxia-inducible factor 1 (HIF-1) and the glucose-responsive transcription factors at the GlcPKRE. The glucose response element (GlcRE) also functioned as a hypoxia response element and, vice versa, a hypoxia-responsive element was functioning as a GlcRE. Thus, our findings implicate that the cross-talk between oxygen and glucose might have a fundamental role in the regulation of several physiological and pathophysiological processes.

Cross-Talk between the Signals Hypoxia and Glucose at the Glucose Response Element of the L-Type Pyruvate Kinase Gene

The signals oxygen and glucose play an important role in metabolism, angiogenesis, tumorigenesis, and embryonic development. Little is known about an interaction of these two signals. We demonstrate here the cross-talk between oxygen and glucose in the regulation of L-type pyruvate kinase (L-PK) gene expression in the liver. In the liver the periportal to perivenous drop in O2 tension was proposed to be an endocrine key regulator for the zonated gene expression. In primary rat hepatocyte cultures the expression of the L-PK gene on mRNA and on protein level was induced by venous pO2, whereas its glucose-dependent induction occurred predominantly under arterial pO2. It was shown by transient transfection of L-PK promoter luciferase and glucose response element (GlcPKRE) SV40 promoter luciferase gene constructs that the modulation by O2 of the glucose-dependent induction occurred at the GlcPKRE in the L-PK gene promoter. The reduction of the glucose-dependent induction of the L-PK gene expression under venous pO2 appeared to be mediated via an interference between hypoxia inducible factor-1 (HIF-1) and upstream stimulating factor at the GlcPKRE. The glucose response element also functioned as an hypoxia response element which was confirmed in cotransfection assays with GlcPKRE luciferase gene constructs and HIF-1α expression vectors. Furthermore, it was found by gel shift and supershift assay that HIF-1α and USF-1 or USF-2 could bind to the GlcPKRE. Our findings implicate that the cross-talk between oxygen and glucose might have a fundamental role in the regulation of several physiological and pathophysiological processes.

Cross-Talk between the Signals Hypoxia and Glucose at the Glucose Response Element of the L-Type Pyruvate Kinase Gene*

The signals oxygen and glucose play an important role in metabolism, angiogenesis, tumorigenesis, and embryonic development. Little is known about an interaction of these two signals. We demonstrate here the cross-talk between oxygen and glucose in the regulation of L-type pyruvate kinase (L-PK) gene expression in the liver. In the liver the periportal to perivenous drop in O(2) tension was proposed to be an endocrine key regulator for the zonated gene expression. In primary rat hepatocyte cultures the expression of the L-PK gene on mRNA and on protein level was induced by venous pO(2), whereas its glucose-dependent induction occurred predominantly under arterial pO(2). It was shown by transient transfection of L-PK promoter luciferase and glucose response element (Glc(PK)RE) SV40 promoter luciferase gene constructs that the modulation by O(2) of the glucose-dependent induction occurred at the Glc(PK)RE in the L-PK gene promoter. The reduction of the glucose-dependent induction of the L-PK gene expression under venous pO(2) appeared to be mediated via an interference between hypoxia inducible factor-1 (HIF-1) and upstream stimulating factor at the Glc(PK)RE. The glucose response element also functioned as an hypoxia response element which was confirmed in cotransfection assays with Glc(PK)RE luciferase gene constructs and HIF-1alpha expression vectors. Furthermore, it was found by gel shift and supershift assay that HIF-1alpha and USF-1 or USF-2 could bind to the Glc(PK)RE. Our findings implicate that the cross-talk between oxygen and glucose might have a fundamental role in the regulation of several physiological and pathophysiological processes.

Negative cyclic AMP response elements in the promoter of the L-type pyruvate kinase gene

L-type pyruvate kinase gene expression is modulated by hormonal and nutritional conditions. Here, we show by transient transfections in hepatocytes in primary culture that both the glucose response element and the contiguous hepatocyte nuclear factor 4 (HNF4) binding site (L3) of the promoter were negative cyclic AMP (cAMP) response elements and that cAMP-dependent inhibition through L3 requires HNF4 binding. Another HNF4 binding site-dependent construct was also inhibited by cAMP. However, HNF4 mutants whose putative PKA-dependent phosphorylation sites have been mutated still conferred cAMP-sensitive transactivation of a L3-dependent reporter gene. Overexpression of the CREB binding protein (CBP) increased the HNF4-dependent transactivation but this effect remained sensitive to cAMP inhibition.

Insulin and cyclic AMP act at different levels on transcription of the L-type pyruvate kinase gene

We previously demonstrated that, in hepatocytes in primary culture, the role of insulin on induction of L-type pyruvate kinase (L-PK) gene expression was mainly to induce glucokinase synthesis, needed for glucose phosphorylation to glucose 6-phosphate. However, we show here that when hepatocytes have been isolated from rats starved for 72 h, glucose and constitutive glucokinase expression was not sufficient to fully stimulate the L-PK promoter, low insulin concentrations being still required. In addition, activation remains sensitive to cAMP inhibition, but cannot be reproduced in the absence of insulin by a competitive cAMP antagonist. We propose that both insulin and cAMP act on expression of the L-PK gene at, at least, two levels: positive and negative regulation of glucokinase gene expression, and more downstream levels.

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.

Diet differentially regulates glucokinase and L-type pyruvate kinase gene expression in rat liver.

The regulation of gene expression of glucokinase (GK) and L-type pyruvate kinase (L-PK) in rat liver was investigated and compared with the previously reported regulation of lipogenic enzymes. Experiments were conducted in which the time courses and responses to diet quantity of mRNA concentrations and enzyme activities after refeeding a carbohydrate/protein diet (CP) to food-deprived rats were measured. The effects of dietary nutrients on the gene expression were investigated in rats refed either the CP diet, a carbohydrate diet without protein (C), a protein diet without carbohydrate (P), or a carbohydrate/protein/corn oil diet (CPF). The effects of the CPF diet on the gene expression after insulin treatment to diabetic rats were also investigated. After refeeding the CP diet, GK mRNA concentration and enzyme activity reached maximum levels in 2 h and 16-24 h, respectively, whereas those of L-PK peaked in 16 h and 48 h, respectively, similar timecourse to lipogenic enzymes. Moreover, GK mRNA concentrations were maximal in rats fed 20% of the ad libitum diet intake, and L-PK mRNA concentrations, like lipogenic enzyme mRNA, were maximal in rats fed approximately 50% of ad libitum intake. GK mRNA concentrations were significantly increased in parallel with an increase in plasma insulin and glucose concentrations. GK and L-PK mRNA and enzyme levels in rats fed the C diet were comparably induced to the levels in those fed the CP diet. L-PK mRNA induction by the CP diet was significantly reduced by dietary polyunsaturated fatty acids (CPF diet), whereas the GK mRNA induction was not significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional Characterization of the L-Type Pyruvate Kinase Gene Glucose Response Complex

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

Functional characterization of the L-type pyruvate kinase gene glucose response complex.

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

Elements responsible for hormonal control and tissue specificity of L-type pyruvate kinase gene expression in transgenic mice.

L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway specifically expressed in the liver and, to a lesser degree, in the small intestine and kidney. One important characteristic of L-PK gene expression is its strong activation by glucose and insulin and its complete inhibition by fasting or glucagon treatment. Having previously established that the entire rat L-PK gene plus 3.2 kbp of 5'-flanking region functions in mice in a tissue-specific and hormonally regulated manner, various deletions of these 3.2 kbp of 5'-flanking regions were tested in transgenic animals to map the cis-acting elements involved in transcriptional gene regulation. Our experiments indicate that the proximal region between -183 and +11 confers tissue specificity and contains all the information necessary for dietary and hormonal control of L-PK gene expression in vivo. We found, however, that the transcriptional activity generated by this proximal promoter fragment can be modulated by distal sequences in a tissue-specific manner. (i) Sequences between bp -183 and -392 seem to play a dual role in the liver and small intestine; they induce L-PK expression in the liver but repress it in the small intestine. (ii) Sequences from bp -392 up to -1170 do not seem to have any additional effect on promoter activity. (iii) Between bp -1170 and -2080, we found a putative extinguisher whose transcriptional inhibitory effect is much more marked in the small intestine than in the liver. (iv) Finally, between bp -2080 and -3200, we identified an activating sequence required for full expression of the gene in the liver.

Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice

New hepatocyte-like cell lines (mhAT) were derived from the liver of a transgenic mouse expressing SV40 early genes under the direction of the liver-specific antithrombin III gene promoter (ATIII-TSV40). Their differentiated phenotypes were improved and stabilized by the use of liver-specific growth media (arginine-free, glucose-free, or low-fructose/glucose-free medium). The best differentiated lines display a very high level of albumin, transferrin, and L-type pyruvate kinase (L-PK) gene expression that is comparable to that observed in the mouse liver. Abundance of the aldolase B and phosphoenolpyruvate carboxykinase (PEPCK) transcripts varied from 5 to 35% of the in vivo concentrations while abundance of the alpha-fetoprotein and phenylalanine hydroxylase transcripts remained very low. Hormonal (cAMP and insulin) and nutritional (glucose) gene controls of PEPCK and L-PK were, at least partially, conserved. mhAT cells are readily transfectable by the calcium phosphate coprecipitation technique and exhibit a liver-specific pattern of expression of exogenous genes. Thus, mhAT cells seem suitable for the analysis of the regulatory regions involved in the tissue-specific transcription of genes. This work demonstrates, therefore, the great efficiency of targeted carcinogenesis in transgenic mice to create new differentiated cell lines. The availability of various lines of liver-specific cells with different phenotypes will constitute useful tools to establish correlations between expression of trans-acting factors and control of the phenotype.

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.

Regulation of L-Type Pyruvate Kinase Gene Expression by Dietary Fructose in Normal and Diabetic Rats1

Previous studies have shown that dietary fructose stimulates expression of the L-type pyruvate kinase gene mainly at the post-transcriptional level in diabetic liver, and that this effect may be mediated by a metabolite common to both fructose and glycerol. In the present work, we carried out further studies on the mechanism of fructose induction of L-type isozyme mRNA in the liver, kidney and small intestine of rats. The L-type isozyme mRNA in the kidney of normal and diabetic rats was increased by dietary fructose, the time course of the increase being similar to that observed in the small intestine and liver. The mRNA was not induced in the kidney by dietary glucose or insulin. Glycerol was also a potent inducer of the mRNA in the kidney and liver, but not in the small intestine. These results show that fructose and glycerol induced increase in the mRNA only in organs in which they were metabolized, and thus support the metabolite hypothesis. No other carbohydrates tested increased the level of mRNA in these tissues, except glucose, which increased the level in the small intestine. Thus a molecule that increase the mRNA level may accumulate significantly during metabolism of only certain carbohydrates. Dietary fructose or glycerol slightly stimulated transcription of the gene for the L-type isozyme in diabetic liver, and also in normal and diabetic kidney, but the magnitudes of increase were much lower than those of the mRNA, confirming our previous findings described above. On the other hand, dietary fructose caused marked stimulation of gene transcription in normal rat liver, although the magnitude of its induction of the mRNA was similar to that in diabetic liver. Insulin treatment of fructose-fed diabetic rats also caused a marked increase in the gene transcription without any concomitant change in the mRNA level. Thus, the mechanism of fructose induction of the L-type pyruvate kinase in diabetic liver, which is similar to that found in the kidney, is different from that in normal liver, and this difference is attributable to the difference in the level of insulin.

In vivo regulation of glycolytic and gluconeogenic enzyme gene expression in newborn rat liver.

Glucagon and its second messenger, cAMP, are known to rapidly block expression of the L-type pyruvate kinase gene and to stimulate expression of phosphoenolpyruvate (PEP) carboxykinase gene in the liver in vivo. The respective roles, however, of hyperglucagonemia, insulinopenia, and carbohydrate deprivation in the inhibition of L-type pyruvate kinase gene expression during fasting are poorly understood. In addition, the long-term effects of physiological hyperglucagonemia on expression of the two genes are not known. In this study, we investigate the effects of long-term physiological hyperglucagonemia and insulinopenia induced by suckling (which provides a high-fat, low-carbohydrate diet) on expression of the two genes in the liver of normal newborn rats. We show that transcription of the L-type pyruvate kinase gene is inhibited at birth and remains low during the whole suckling period, whereas transcription of the PEP carboxykinase gene is maximal in the neonate, and then decreases despite very high levels of plasma glucagon during suckling. In contrast to the adult, however, in which L-type pyruvate kinase gene expression in the liver is blocked by cAMP and stimulated by carbohydrates, the regulation of L-type pyruvate kinase gene expression in the newborn undergoes a developmental maturation: the inhibitory effect of glucagon is never complete in developing rat liver and the stimulatory effect of glucose could not be detected during suckling, due to either hyperglucagonemia, immaturity of the gene regulatory system, or both.

Differential effects of glucose and fructose on liver L-type pyruvate kinase gene expression in vivo.

Dietary carbohydrates are known to stimulate L-type pyruvate kinase gene expression at the transcriptional level in the liver. However, the short-term effects, the time course, and the mechanism of the gene activation by elemental hexoses in normal fasted rats remain unknown. In the present study, both glucose and fructose were found to stimulate the gene expression at the transcriptional level in liver. However, the kinetics and the extent of the mRNA induction differed according to the carbohydrate given. Fructose stimulated early (2-4 h) and transiently the gene transcription, the RNA precursor, and the mRNA accumulation in 48-h-fasted rats while maximum stimulation of the RNA synthesis by glucose was delayed until the 12th h of refeeding, despite an early rise of plasma insulin. In contrast, insulin release was not required for fructose to trigger the gene transcription, nor did the high cyclic AMP levels in fasted rat liver prevent RNA synthesis by fructose. The agent(s) operating early in fructose-fed animals might be powerful enough to not require insulin for gene activation and to balance the inhibitory action of glucagon in the liver.

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.

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.

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.