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Regulation of the Glucose-6-phosphatase Gene by Glucose Occurs by Transcriptional and Post-transcriptional Mechanisms

2001; Elsevier BV; Volume: 276; Issue: 6 Linguagem: Inglês

10.1074/jbc.m007939200

ISSN

1083-351X

Autores

Duna Massillon,

Tópico(s)

Amino Acid Enzymes and Metabolism

Resumo

To understand how glucose regulates the expression of the glucose-6-phosphatase gene, the effect of glucose was studied in primary cultures of rat hepatocytes. Glucose-6-phosphatase mRNA levels increased about 10-fold when hepatocytes were incubated with 20 mm glucose. The rate of transcription of the glucose-6-phosphatase gene increased about 3-fold in hepatocytes incubated with glucose. The half-life of glucose-6-phosphatase mRNA was estimated to be 90 min in the absence of glucose and 3 h in its presence. Inhibition of the oxidative and the nonoxidative branches of the pentose phosphate pathway blocked the stimulation of glucose-6-phosphatase expression by glucose but not by xylitol or carbohydrates that enter the glycolytic/gluconeogenic pathways at the level of the triose phosphates. These results indicate that (i) the glucose induction of the mRNA for the catalytic unit of glucose-6-phosphatase occurs by transcriptional and post-transcriptional mechanisms and that (ii) xylitol and glucose increase the expression of this gene through different signaling pathways. To understand how glucose regulates the expression of the glucose-6-phosphatase gene, the effect of glucose was studied in primary cultures of rat hepatocytes. Glucose-6-phosphatase mRNA levels increased about 10-fold when hepatocytes were incubated with 20 mm glucose. The rate of transcription of the glucose-6-phosphatase gene increased about 3-fold in hepatocytes incubated with glucose. The half-life of glucose-6-phosphatase mRNA was estimated to be 90 min in the absence of glucose and 3 h in its presence. Inhibition of the oxidative and the nonoxidative branches of the pentose phosphate pathway blocked the stimulation of glucose-6-phosphatase expression by glucose but not by xylitol or carbohydrates that enter the glycolytic/gluconeogenic pathways at the level of the triose phosphates. These results indicate that (i) the glucose induction of the mRNA for the catalytic unit of glucose-6-phosphatase occurs by transcriptional and post-transcriptional mechanisms and that (ii) xylitol and glucose increase the expression of this gene through different signaling pathways. glucose-6-phosphatase 6-aminonicotinamide glucose 6-phosphate glucose-6-phosphate dehydrogenase phosphoenolpyruvate carboxykinase phosphogluconate dehydrogenase 4-morpholinepropanesulfonic acid untranslated region AU-rich element Glc-6-Pase1 (EC 3.1.3.9) is a multicomponent protein complex comprising catalytic and transporting entities (1Sukalski K.A. Nordlie R.C. Adv. Enzymol. 1989; 62: 93-117PubMed Google Scholar, 2Nordlie R.C. Bode A.M. Foster J.D. Proc. Soc. Exp. Biol. Med. 1993; 203: 274-285Crossref PubMed Scopus (46) Google Scholar, 3Arion W.J. Lange A.J. Walls H. Ballas L. J. Biol. Chem. 1980; 255: 10396-10406Abstract Full Text PDF PubMed Google Scholar, 4Burchell A. FASEB J. 1990; 4: 2978-2988Crossref PubMed Scopus (92) Google Scholar, 5van de Werve G. J. Biol. Chem. 1989; 264: 6033-6036Abstract Full Text PDF PubMed Google Scholar). The complex is tightly associated with the endoplasmic reticulum, and the enzymatic component catalyzes the hydrolysis of glucose 6-phosphate to glucose, a final common step to both the pathways of glycogenolysis and gluconeogenesis. Hepatic Glc-6-Pase activity is effectively regulated by hormonal and nutritional status. For example, fasting and hormones that increase cAMP concentration stimulate its gene expression while re-feeding and insulin decrease it (1Sukalski K.A. Nordlie R.C. Adv. Enzymol. 1989; 62: 93-117PubMed Google Scholar, 2Nordlie R.C. Bode A.M. Foster J.D. Proc. Soc. Exp. Biol. Med. 1993; 203: 274-285Crossref PubMed Scopus (46) Google Scholar, 6Rossetti L. Giaccari A. Barzilai N. Howards K. Sebel G. Hu M. J. Clin. Invest. 1993; 92: 1126-1134Crossref PubMed Scopus (176) Google Scholar, 7Gardner L. Liu Z. Barrett E. Diabetes. 1993; 42: 1614-1620Crossref PubMed Scopus (38) Google Scholar, 8Lange A.J. Argaud D. El-Maghrabi M.R. Pan W. Maitra S.R. Pilkis S.J. Biochem. Biophys. Res. Commun. 1994; 201: 302-309Crossref PubMed Scopus (116) Google Scholar, 9Liu Z. Barrett E.J. Dalkin A.C. Zwart A.D. Chou J.Y. Biochem. Biophys. Res. Commun. 1994; 205: 680-686Crossref PubMed Scopus (95) Google Scholar, 10Barzilai N. Massillon D. Rossetti L. Biochem. J. 1995; 310: 819-826Crossref PubMed Scopus (20) Google Scholar, 11Massillon D. Barzilai N. Chen W. Hu M. Rossetti L. J. Biol. Chem. 1996; 271: 9871-9874Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 12Massillon D. Chen W. Barzilai N. Prus-Wertheimer D. Hawkins M. Liu R. Taub R. Rossetti L. J. Biol. Chem. 1998; 273: 228-234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).The expression of the genes for several other proteins is regulated by glucose; these include genes for L-type pyruvate kinase (13Munnich A. Marie J. Reach G. Vaulont S. Simon M. Kahn A. J. Biol. Chem. 1984; 259: 10228-10231Abstract Full Text PDF PubMed Google Scholar, 14Vaulont S. Munnich A. Decaux J.F. Kahn A. J. Biol. Chem. 1986; 261: 7621-7625Abstract Full Text PDF PubMed Google Scholar), fatty-acid synthase (15Foufelle F. Girard J. Ferre P. Adv. Enzyme Regul. 1992; 36: 199-226Crossref Scopus (71) Google Scholar, 16Prip-Buus C. Perdereau D. Foufelle F. Maury J. Ferre P. Girard J. Eur. J. Biochem. 1995; 230: 309-315Crossref PubMed Scopus (88) Google Scholar), PEPCK (17Kahn R.C. Lauris V. Koch S. Crettaz M. Granner D.K. Mol. Endocrinol. 1989; 3: 840-845Crossref PubMed Scopus (54) Google Scholar, 18Foufelle F. Gouhot B. Perdereau D. Girard J. Ferre P. Eur. J. Biochem. 1994; 223: 893-900Crossref PubMed Scopus (28) Google Scholar), and the type 2 glucose transporter (GLUT-2) (19Burcelin R. Eddouks M. Kande J. Girard J. Biochem. J. 1992; 288: 675-679Crossref PubMed Scopus (75) Google Scholar, 20Rencurel F. Waeber G. Antoine B. Rocchiccioli F. Maulard P. Girard J. Leturque A. Biochem. J. 1996; 314: 903-909Crossref PubMed Scopus (95) Google Scholar). The mechanism by which glucose regulates the expression of these genes remains largely unknown. Recently, Kahn and colleagues (21Kahn A. Biochimie (Paris). 1997; 79: 113-118Crossref PubMed Scopus (53) Google Scholar, 22Mitanchez D. Doiron B. Chen R. Kahn A. Endocr. Rev. 1997; 18: 520-540PubMed Google Scholar) have proposed a signaling pathway model to explain the molecular mechanism by which glucose regulates the expression of the L-type pyruvate kinase. This model consists of the following details: (i) the presence of glucose is sensed in the cell; (ii) this information is transduced by intracellular messengers; (iii) a second messenger, presumably xylulose 5-phosphate, rises and then modulates the activity of protein kinase and protein phosphatase involved in a cascade of phosphorylation/dephosphorylation. This cascade then leads to a modification of the phosphorylation state of the glucose-responsive complex, followed by an increase in the transcriptional rate of the target gene. In this model, the presence of an active glucokinase that phosphorylates glucose to glucose 6-phosphate (Glc-6-P) is paramount.The molecular mechanism by which glucose regulates the expression of the Glc-6-Pase gene is currently unknown. Here we show that glucose regulation of the expression of this gene involves metabolism of glucose through the glycolytic pathway, transcriptional activation of the Glc-6-Pase gene promoter, and a decrease in the degradation of Glc-6-Pase mRNA.DISCUSSIONThe molecular mechanisms underlying glucose-induced regulation of the Glc-6-Pase gene is not clearly understood. Here we present evidence that stimulation of Glc-6-Pase gene expression by glucose involves both transcriptional and post-transcriptional components. The specific DNA sequence(s) responsible for the transcriptional response remains to be established. The half-life of Glc-6-Pase mRNA was found to be 90 min. The fact that glucose prolongs this half-life suggests glucose-induced stabilization of the Glc-6-Pase mRNA. New protein synthesis does not seem to be necessary for glucose to induce Glc-6-Pase mRNA. This would argue against a mechanism whereby glucose stimulated the synthesis of a protein factor(s) that bind to the mRNA and thereby prevent its degradation. The stabilization of the Glc-6-Pase mRNA by glucose does not depend on the ability of this mRNA to be translated, since the 2-fold increase in the level of Glc-6-Pase protein was much lower than the increase in its mRNA. It has been extensively documented that mRNAs whose 3′-UTRs contain an AU-rich element (AURE) and/or an oligonucleotide (U) tend to be unstable (36Caput D. Beutle R.B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1208) Google Scholar). Such AURE in the 3′-UTR had been found in transcripts such as c-fos and c-myc (37Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (399) Google Scholar, 38You Y. Chen C.-Y. Shyu A.-B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar) that are known to have a short half-life. Their significance in mRNA turnover has been demonstrated in vivo and in vitro (38You Y. Chen C.-Y. Shyu A.-B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar, 39Cleveland D.W. Yen T.J. New Biol. 1989; 1: 121-126PubMed Google Scholar, 40Bohjanen P.R. Petrymiak B. June C.H. Thompson C.B. Lindsten T. Mol. Cell. Biol. 1991; 11: 3288-3295Crossref PubMed Scopus (233) Google Scholar, 41Bohjanen P.R. Petrymiak B. June C.H. Thompson C.B. Lindsten T. J. Biol. Chem. 1992; 267: 6302-6309Abstract Full Text PDF PubMed Google Scholar). The 3′-UTR of the Glc-6-Pase gene contains a few AUREs. Whether these AUREs play any role in Glc-6-Pase mRNA stability is not known.One unresolved question about the glucose-induced accumulation of Glc-6-Pase mRNA is whether this effect requires glucose metabolism through the pentose phosphate pathway. If this pathway is important for the effect, then suppressing it should be expected to prevent the accumulation of Glc-6-Pase mRNA. Glucose-induced accumulation of the mRNA for Glc-6-Pase was indeed prevented when hepatocytes were incubated with 0.2 mm 6-AN. When metabolized to 6-amino-NAD and 6-amino-NADP, this nucleotide behaves as a competitive inhibitor of NAD(P+)-requiring dehydrogenases that would include G6PDH, 6-PGDH, and glutathione reductase (32Downs S.M. Humpherson P.G. Leese H.J. Biol. Reprod. 1998; 58: 1084-1094Crossref PubMed Scopus (120) Google Scholar, 33Street J.C. Mahmood U. Ballon D. Alfieri A.A. Koutcher J.A. J. Biol. Chem. 1996; 271: 4113-4119Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 42Carmona A. Freedland R.A. Int. J. Biochem. 1990; 22: 595-599Crossref PubMed Scopus (10) Google Scholar, 43Kohler E. Barrach H.-J. Neubert D. FEBS Lett. 1970; 6: 225-228Crossref PubMed Scopus (118) Google Scholar, 44Street J.C. Alfieri A.A. Koutcher J.A. Cancer Res. 1997; 57: 3956-3962PubMed Google Scholar, 45Sudo J.-I. Ishihara A. Tanabe T. Jpn. J. Pharmacol. 1984; 36: 491-498Crossref PubMed Scopus (1) Google Scholar, 46Hashizume K. Onaya T. Sato A. Endocrinology. 1975; 97: 962-968Crossref PubMed Scopus (16) Google Scholar, 48Coper H. Neubert D. Biochim. Biophys. Acta. 1964; 89: 23-32PubMed Google Scholar). 6-Amino-NADP is an extremely potent competitive inhibitor of 6-PGDH, and concentrations that do not affect G6PDH may completely block 6-PGDH (43Kohler E. Barrach H.-J. Neubert D. FEBS Lett. 1970; 6: 225-228Crossref PubMed Scopus (118) Google Scholar). On the other hand, no inhibition of NAD-dependent enzymes results from the use of this compound (48Coper H. Neubert D. Biochim. Biophys. Acta. 1964; 89: 23-32PubMed Google Scholar). The inhibition of glycolysis from glucose by 6-AN makes it difficult to interpret the results using glucose as an energy source. Nevertheless, this compound is very valuable to study the effect of substrates such as fructose and glycerol that enter glycolysis beyond the formation of fructose 6-phosphate.When both the pentose phosphate pathway and phosphoglucoisomerase were inhibited with 6-AN and oxythiamine, stimulation of Glc-6-Pase gene expression by fructose or glycerol was not affected. These two substrates enter the glycolytic pathway at the level of triose phosphate(s), and the fact that the nonoxidative branch of the pentose phosphate pathway was blocked suggests that the pentose phosphate pathway is not required for the glucose effect on the Glc-6-Pase gene. In contrast to glucose, xylitol induced Glc-6-Pase mRNA even in the presence of inhibitors of the pentose phosphate pathway. Taken together, these results support the idea that xylitol and glucose signal through different pathways.The signaling pathway from glucose to the DNA sequences termed carbohydrate responsive element (ChoRE/GIRE) is not totally characterized. Phosphorylation/dephosphorylation of transcription factors have been implicated in the glucose responsiveness of many genes. Xylulose 5-phosphate, produced by the pentose phosphate pathway, has been suggested as a secondary messenger in sensing glucose concentration in the hepatocyte. This proposal stems from the fact that xylitol, a precursor of xylulose 5-phosphate, is able to stimulate the expression of a number of genes, both in vivo (12Massillon D. Chen W. Barzilai N. Prus-Wertheimer D. Hawkins M. Liu R. Taub R. Rossetti L. J. Biol. Chem. 1998; 273: 228-234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and in cultured cells (26Doiron B. Cuif M.-H. Chen R. Kahn A. J. Biol. Chem. 1996; 271: 5321-5324Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 28Mourrieras F. Foufelle F. Foretz M. Morin J. Bouche S. Ferre P. Biochem. J. 1997; 326: 345-349Crossref PubMed Scopus (68) Google Scholar). Xylulose 5-phosphate has been shown to activate the phosphatase 2A-mediated dephosphorylation (49Nishimura M. Uyeda K. J. Biol. Chem. 1995; 270: 26341-26346Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 50Liu Y.Q. Uyeda K. J. Biol. Chem. 1996; 271: 8824-8830Abstract Full Text PDF PubMed Scopus (31) Google Scholar) of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase and to decrease the activity of protein kinase A. The same phosphatase is also involved in the dephosphorylation of the transcription factor Sp1 (51Alberts A.S. Deng T. Lin A. Meinkoth J.L. Schonthal A. Mumby M.C. Karin M. Feramisco J.R. Mol. Cell. Biol. 1993; 13: 2104-2112Crossref PubMed Scopus (67) Google Scholar,52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Xylulose 5-phosphate-activated phosphatase is also activated by glucose or a glucose metabolite (50Liu Y.Q. Uyeda K. J. Biol. Chem. 1996; 271: 8824-8830Abstract Full Text PDF PubMed Scopus (31) Google Scholar). Furthermore, glucose-induced transcription of the acetyl-CoA carboxylase gene is mediated by the transcription factor Sp1 (52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). This stimulation is prevented by okadaic acid, an inhibitor of protein phosphatase types 1 and 2A (52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). On the other hand, okadaic acid only partially inhibits the action of glucose and xylitol to stimulate the fructose-6-phosphate,2-kinase:fructose-2,6-biphosphatase (53Dupriez V.J. Rousseau G.G. Mol. Cell. Biol. 1997; 16: 1075-1085Google Scholar). These results suggest that either dephosphorylation of Sp1 is not universal in the glucose signaling pathway or that glucose signaling also involves other transcription factors such as the upstreamstimulatory factor proteins (54Lefrancois-Martinez A.-M Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: 2640-2643Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). These upstream stimulatory factor proteins were among the first transcription factors suggested to link glucose action to the ChoRE/GIRE (54Lefrancois-Martinez A.-M Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: 2640-2643Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 55Vaulont S. Kahn A. FASEB J. 1994; 8: 28-35Crossref PubMed Scopus (174) Google Scholar). The involvement of protein phosphatase(s) in the modulation of transcription factors involved in glucose signaling is worth exploring further.The metabolic consequences of increased Glc-6-Pase gene expression by glucose are not known. More than 25 years ago, Nordlie and colleagues (47Nordlie R.C. Hanson R.W. Mehlman M.A. Gluconeogenesis, Its Regulation in Mammalian Species. John Wiley & Sons, New York1976: 93-152Google Scholar) proposed the controversial idea that Glc-6-Pase, in the presence of high glucose levels, might act as a phosphotransferase that uses carbamoyl phosphate as the phosphate donor. Whether Glc-6-Pase plays any role in glucose phosphorylation is debatable. Nonetheless, the paradoxical induction of Glc-6-Pase gene by glucose points toward a potential role of this enzyme in the removal of glucose from the circulation. Glucose stimulation might also be needed to make sure that the enzyme does not disappear when the cell shifts from fed to starved conditions. One speculation is that glucose stimulation of Glc-6-Pase gene may take place in vivo when glucose cycling is needed following hyperglycemia. Glc-6-Pase is a very complex protein with different protein components that are not yet well characterized. Glc-6-Pase1 (EC 3.1.3.9) is a multicomponent protein complex comprising catalytic and transporting entities (1Sukalski K.A. Nordlie R.C. Adv. Enzymol. 1989; 62: 93-117PubMed Google Scholar, 2Nordlie R.C. Bode A.M. Foster J.D. Proc. Soc. Exp. Biol. Med. 1993; 203: 274-285Crossref PubMed Scopus (46) Google Scholar, 3Arion W.J. Lange A.J. Walls H. Ballas L. J. Biol. Chem. 1980; 255: 10396-10406Abstract Full Text PDF PubMed Google Scholar, 4Burchell A. FASEB J. 1990; 4: 2978-2988Crossref PubMed Scopus (92) Google Scholar, 5van de Werve G. J. Biol. Chem. 1989; 264: 6033-6036Abstract Full Text PDF PubMed Google Scholar). The complex is tightly associated with the endoplasmic reticulum, and the enzymatic component catalyzes the hydrolysis of glucose 6-phosphate to glucose, a final common step to both the pathways of glycogenolysis and gluconeogenesis. Hepatic Glc-6-Pase activity is effectively regulated by hormonal and nutritional status. For example, fasting and hormones that increase cAMP concentration stimulate its gene expression while re-feeding and insulin decrease it (1Sukalski K.A. Nordlie R.C. Adv. Enzymol. 1989; 62: 93-117PubMed Google Scholar, 2Nordlie R.C. Bode A.M. Foster J.D. Proc. Soc. Exp. Biol. Med. 1993; 203: 274-285Crossref PubMed Scopus (46) Google Scholar, 6Rossetti L. Giaccari A. Barzilai N. Howards K. Sebel G. Hu M. J. Clin. Invest. 1993; 92: 1126-1134Crossref PubMed Scopus (176) Google Scholar, 7Gardner L. Liu Z. Barrett E. Diabetes. 1993; 42: 1614-1620Crossref PubMed Scopus (38) Google Scholar, 8Lange A.J. Argaud D. El-Maghrabi M.R. Pan W. Maitra S.R. Pilkis S.J. Biochem. Biophys. Res. Commun. 1994; 201: 302-309Crossref PubMed Scopus (116) Google Scholar, 9Liu Z. Barrett E.J. Dalkin A.C. Zwart A.D. Chou J.Y. Biochem. Biophys. Res. Commun. 1994; 205: 680-686Crossref PubMed Scopus (95) Google Scholar, 10Barzilai N. Massillon D. Rossetti L. Biochem. J. 1995; 310: 819-826Crossref PubMed Scopus (20) Google Scholar, 11Massillon D. Barzilai N. Chen W. Hu M. Rossetti L. J. Biol. Chem. 1996; 271: 9871-9874Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 12Massillon D. Chen W. Barzilai N. Prus-Wertheimer D. Hawkins M. Liu R. Taub R. Rossetti L. J. Biol. Chem. 1998; 273: 228-234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The expression of the genes for several other proteins is regulated by glucose; these include genes for L-type pyruvate kinase (13Munnich A. Marie J. Reach G. Vaulont S. Simon M. Kahn A. J. Biol. Chem. 1984; 259: 10228-10231Abstract Full Text PDF PubMed Google Scholar, 14Vaulont S. Munnich A. Decaux J.F. Kahn A. J. Biol. Chem. 1986; 261: 7621-7625Abstract Full Text PDF PubMed Google Scholar), fatty-acid synthase (15Foufelle F. Girard J. Ferre P. Adv. Enzyme Regul. 1992; 36: 199-226Crossref Scopus (71) Google Scholar, 16Prip-Buus C. Perdereau D. Foufelle F. Maury J. Ferre P. Girard J. Eur. J. Biochem. 1995; 230: 309-315Crossref PubMed Scopus (88) Google Scholar), PEPCK (17Kahn R.C. Lauris V. Koch S. Crettaz M. Granner D.K. Mol. Endocrinol. 1989; 3: 840-845Crossref PubMed Scopus (54) Google Scholar, 18Foufelle F. Gouhot B. Perdereau D. Girard J. Ferre P. Eur. J. Biochem. 1994; 223: 893-900Crossref PubMed Scopus (28) Google Scholar), and the type 2 glucose transporter (GLUT-2) (19Burcelin R. Eddouks M. Kande J. Girard J. Biochem. J. 1992; 288: 675-679Crossref PubMed Scopus (75) Google Scholar, 20Rencurel F. Waeber G. Antoine B. Rocchiccioli F. Maulard P. Girard J. Leturque A. Biochem. J. 1996; 314: 903-909Crossref PubMed Scopus (95) Google Scholar). The mechanism by which glucose regulates the expression of these genes remains largely unknown. Recently, Kahn and colleagues (21Kahn A. Biochimie (Paris). 1997; 79: 113-118Crossref PubMed Scopus (53) Google Scholar, 22Mitanchez D. Doiron B. Chen R. Kahn A. Endocr. Rev. 1997; 18: 520-540PubMed Google Scholar) have proposed a signaling pathway model to explain the molecular mechanism by which glucose regulates the expression of the L-type pyruvate kinase. This model consists of the following details: (i) the presence of glucose is sensed in the cell; (ii) this information is transduced by intracellular messengers; (iii) a second messenger, presumably xylulose 5-phosphate, rises and then modulates the activity of protein kinase and protein phosphatase involved in a cascade of phosphorylation/dephosphorylation. This cascade then leads to a modification of the phosphorylation state of the glucose-responsive complex, followed by an increase in the transcriptional rate of the target gene. In this model, the presence of an active glucokinase that phosphorylates glucose to glucose 6-phosphate (Glc-6-P) is paramount. The molecular mechanism by which glucose regulates the expression of the Glc-6-Pase gene is currently unknown. Here we show that glucose regulation of the expression of this gene involves metabolism of glucose through the glycolytic pathway, transcriptional activation of the Glc-6-Pase gene promoter, and a decrease in the degradation of Glc-6-Pase mRNA. DISCUSSIONThe molecular mechanisms underlying glucose-induced regulation of the Glc-6-Pase gene is not clearly understood. Here we present evidence that stimulation of Glc-6-Pase gene expression by glucose involves both transcriptional and post-transcriptional components. The specific DNA sequence(s) responsible for the transcriptional response remains to be established. The half-life of Glc-6-Pase mRNA was found to be 90 min. The fact that glucose prolongs this half-life suggests glucose-induced stabilization of the Glc-6-Pase mRNA. New protein synthesis does not seem to be necessary for glucose to induce Glc-6-Pase mRNA. This would argue against a mechanism whereby glucose stimulated the synthesis of a protein factor(s) that bind to the mRNA and thereby prevent its degradation. The stabilization of the Glc-6-Pase mRNA by glucose does not depend on the ability of this mRNA to be translated, since the 2-fold increase in the level of Glc-6-Pase protein was much lower than the increase in its mRNA. It has been extensively documented that mRNAs whose 3′-UTRs contain an AU-rich element (AURE) and/or an oligonucleotide (U) tend to be unstable (36Caput D. Beutle R.B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1208) Google Scholar). Such AURE in the 3′-UTR had been found in transcripts such as c-fos and c-myc (37Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (399) Google Scholar, 38You Y. Chen C.-Y. Shyu A.-B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar) that are known to have a short half-life. Their significance in mRNA turnover has been demonstrated in vivo and in vitro (38You Y. Chen C.-Y. Shyu A.-B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar, 39Cleveland D.W. Yen T.J. New Biol. 1989; 1: 121-126PubMed Google Scholar, 40Bohjanen P.R. Petrymiak B. June C.H. Thompson C.B. Lindsten T. Mol. Cell. Biol. 1991; 11: 3288-3295Crossref PubMed Scopus (233) Google Scholar, 41Bohjanen P.R. Petrymiak B. June C.H. Thompson C.B. Lindsten T. J. Biol. Chem. 1992; 267: 6302-6309Abstract Full Text PDF PubMed Google Scholar). The 3′-UTR of the Glc-6-Pase gene contains a few AUREs. Whether these AUREs play any role in Glc-6-Pase mRNA stability is not known.One unresolved question about the glucose-induced accumulation of Glc-6-Pase mRNA is whether this effect requires glucose metabolism through the pentose phosphate pathway. If this pathway is important for the effect, then suppressing it should be expected to prevent the accumulation of Glc-6-Pase mRNA. Glucose-induced accumulation of the mRNA for Glc-6-Pase was indeed prevented when hepatocytes were incubated with 0.2 mm 6-AN. When metabolized to 6-amino-NAD and 6-amino-NADP, this nucleotide behaves as a competitive inhibitor of NAD(P+)-requiring dehydrogenases that would include G6PDH, 6-PGDH, and glutathione reductase (32Downs S.M. Humpherson P.G. Leese H.J. Biol. Reprod. 1998; 58: 1084-1094Crossref PubMed Scopus (120) Google Scholar, 33Street J.C. Mahmood U. Ballon D. Alfieri A.A. Koutcher J.A. J. Biol. Chem. 1996; 271: 4113-4119Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 42Carmona A. Freedland R.A. Int. J. Biochem. 1990; 22: 595-599Crossref PubMed Scopus (10) Google Scholar, 43Kohler E. Barrach H.-J. Neubert D. FEBS Lett. 1970; 6: 225-228Crossref PubMed Scopus (118) Google Scholar, 44Street J.C. Alfieri A.A. Koutcher J.A. Cancer Res. 1997; 57: 3956-3962PubMed Google Scholar, 45Sudo J.-I. Ishihara A. Tanabe T. Jpn. J. Pharmacol. 1984; 36: 491-498Crossref PubMed Scopus (1) Google Scholar, 46Hashizume K. Onaya T. Sato A. Endocrinology. 1975; 97: 962-968Crossref PubMed Scopus (16) Google Scholar, 48Coper H. Neubert D. Biochim. Biophys. Acta. 1964; 89: 23-32PubMed Google Scholar). 6-Amino-NADP is an extremely potent competitive inhibitor of 6-PGDH, and concentrations that do not affect G6PDH may completely block 6-PGDH (43Kohler E. Barrach H.-J. Neubert D. FEBS Lett. 1970; 6: 225-228Crossref PubMed Scopus (118) Google Scholar). On the other hand, no inhibition of NAD-dependent enzymes results from the use of this compound (48Coper H. Neubert D. Biochim. Biophys. Acta. 1964; 89: 23-32PubMed Google Scholar). The inhibition of glycolysis from glucose by 6-AN makes it difficult to interpret the results using glucose as an energy source. Nevertheless, this compound is very valuable to study the effect of substrates such as fructose and glycerol that enter glycolysis beyond the formation of fructose 6-phosphate.When both the pentose phosphate pathway and phosphoglucoisomerase were inhibited with 6-AN and oxythiamine, stimulation of Glc-6-Pase gene expression by fructose or glycerol was not affected. These two substrates enter the glycolytic pathway at the level of triose phosphate(s), and the fact that the nonoxidative branch of the pentose phosphate pathway was blocked suggests that the pentose phosphate pathway is not required for the glucose effect on the Glc-6-Pase gene. In contrast to glucose, xylitol induced Glc-6-Pase mRNA even in the presence of inhibitors of the pentose phosphate pathway. Taken together, these results support the idea that xylitol and glucose signal through different pathways.The signaling pathway from glucose to the DNA sequences termed carbohydrate responsive element (ChoRE/GIRE) is not totally characterized. Phosphorylation/dephosphorylation of transcription factors have been implicated in the glucose responsiveness of many genes. Xylulose 5-phosphate, produced by the pentose phosphate pathway, has been suggested as a secondary messenger in sensing glucose concentration in the hepatocyte. This proposal stems from the fact that xylitol, a precursor of xylulose 5-phosphate, is able to stimulate the expression of a number of genes, both in vivo (12Massillon D. Chen W. Barzilai N. Prus-Wertheimer D. Hawkins M. Liu R. Taub R. Rossetti L. J. Biol. Chem. 1998; 273: 228-234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and in cultured cells (26Doiron B. Cuif M.-H. Chen R. Kahn A. J. Biol. Chem. 1996; 271: 5321-5324Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 28Mourrieras F. Foufelle F. Foretz M. Morin J. Bouche S. Ferre P. Biochem. J. 1997; 326: 345-349Crossref PubMed Scopus (68) Google Scholar). Xylulose 5-phosphate has been shown to activate the phosphatase 2A-mediated dephosphorylation (49Nishimura M. Uyeda K. J. Biol. Chem. 1995; 270: 26341-26346Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 50Liu Y.Q. Uyeda K. J. Biol. Chem. 1996; 271: 8824-8830Abstract Full Text PDF PubMed Scopus (31) Google Scholar) of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase and to decrease the activity of protein kinase A. The same phosphatase is also involved in the dephosphorylation of the transcription factor Sp1 (51Alberts A.S. Deng T. Lin A. Meinkoth J.L. Schonthal A. Mumby M.C. Karin M. Feramisco J.R. Mol. Cell. Biol. 1993; 13: 2104-2112Crossref PubMed Scopus (67) Google Scholar,52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Xylulose 5-phosphate-activated phosphatase is also activated by glucose or a glucose metabolite (50Liu Y.Q. Uyeda K. J. Biol. Chem. 1996; 271: 8824-8830Abstract Full Text PDF PubMed Scopus (31) Google Scholar). Furthermore, glucose-induced transcription of the acetyl-CoA carboxylase gene is mediated by the transcription factor Sp1 (52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). This stimulation is prevented by okadaic acid, an inhibitor of protein phosphatase types 1 and 2A (52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). On the other hand, okadaic acid only partially inhibits the action of glucose and xylitol to stimulate the fructose-6-phosphate,2-kinase:fructose-2,6-biphosphatase (53Dupriez V.J. Rousseau G.G. Mol. Cell. Biol. 1997; 16: 1075-1085Google Scholar). These results suggest that either dephosphorylation of Sp1 is not universal in the glucose signaling pathway or that glucose signaling also involves other transcription factors such as the upstreamstimulatory factor proteins (54Lefrancois-Martinez A.-M Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: 2640-2643Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). These upstream stimulatory factor proteins were among the first transcription factors suggested to link glucose action to the ChoRE/GIRE (54Lefrancois-Martinez A.-M Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: 2640-2643Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 55Vaulont S. Kahn A. FASEB J. 1994; 8: 28-35Crossref PubMed Scopus (174) Google Scholar). The involvement of protein phosphatase(s) in the modulation of transcription factors involved in glucose signaling is worth exploring further.The metabolic consequences of increased Glc-6-Pase gene expression by glucose are not known. More than 25 years ago, Nordlie and colleagues (47Nordlie R.C. Hanson R.W. Mehlman M.A. Gluconeogenesis, Its Regulation in Mammalian Species. John Wiley & Sons, New York1976: 93-152Google Scholar) proposed the controversial idea that Glc-6-Pase, in the presence of high glucose levels, might act as a phosphotransferase that uses carbamoyl phosphate as the phosphate donor. Whether Glc-6-Pase plays any role in glucose phosphorylation is debatable. Nonetheless, the paradoxical induction of Glc-6-Pase gene by glucose points toward a potential role of this enzyme in the removal of glucose from the circulation. Glucose stimulation might also be needed to make sure that the enzyme does not disappear when the cell shifts from fed to starved conditions. One speculation is that glucose stimulation of Glc-6-Pase gene may take place in vivo when glucose cycling is needed following hyperglycemia. Glc-6-Pase is a very complex protein with different protein components that are not yet well characterized. The molecular mechanisms underlying glucose-induced regulation of the Glc-6-Pase gene is not clearly understood. Here we present evidence that stimulation of Glc-6-Pase gene expression by glucose involves both transcriptional and post-transcriptional components. The specific DNA sequence(s) responsible for the transcriptional response remains to be established. The half-life of Glc-6-Pase mRNA was found to be 90 min. The fact that glucose prolongs this half-life suggests glucose-induced stabilization of the Glc-6-Pase mRNA. New protein synthesis does not seem to be necessary for glucose to induce Glc-6-Pase mRNA. This would argue against a mechanism whereby glucose stimulated the synthesis of a protein factor(s) that bind to the mRNA and thereby prevent its degradation. The stabilization of the Glc-6-Pase mRNA by glucose does not depend on the ability of this mRNA to be translated, since the 2-fold increase in the level of Glc-6-Pase protein was much lower than the increase in its mRNA. It has been extensively documented that mRNAs whose 3′-UTRs contain an AU-rich element (AURE) and/or an oligonucleotide (U) tend to be unstable (36Caput D. Beutle R.B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1208) Google Scholar). Such AURE in the 3′-UTR had been found in transcripts such as c-fos and c-myc (37Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (399) Google Scholar, 38You Y. Chen C.-Y. Shyu A.-B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar) that are known to have a short half-life. Their significance in mRNA turnover has been demonstrated in vivo and in vitro (38You Y. Chen C.-Y. Shyu A.-B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar, 39Cleveland D.W. Yen T.J. New Biol. 1989; 1: 121-126PubMed Google Scholar, 40Bohjanen P.R. Petrymiak B. June C.H. Thompson C.B. Lindsten T. Mol. Cell. Biol. 1991; 11: 3288-3295Crossref PubMed Scopus (233) Google Scholar, 41Bohjanen P.R. Petrymiak B. June C.H. Thompson C.B. Lindsten T. J. Biol. Chem. 1992; 267: 6302-6309Abstract Full Text PDF PubMed Google Scholar). The 3′-UTR of the Glc-6-Pase gene contains a few AUREs. Whether these AUREs play any role in Glc-6-Pase mRNA stability is not known. One unresolved question about the glucose-induced accumulation of Glc-6-Pase mRNA is whether this effect requires glucose metabolism through the pentose phosphate pathway. If this pathway is important for the effect, then suppressing it should be expected to prevent the accumulation of Glc-6-Pase mRNA. Glucose-induced accumulation of the mRNA for Glc-6-Pase was indeed prevented when hepatocytes were incubated with 0.2 mm 6-AN. When metabolized to 6-amino-NAD and 6-amino-NADP, this nucleotide behaves as a competitive inhibitor of NAD(P+)-requiring dehydrogenases that would include G6PDH, 6-PGDH, and glutathione reductase (32Downs S.M. Humpherson P.G. Leese H.J. Biol. Reprod. 1998; 58: 1084-1094Crossref PubMed Scopus (120) Google Scholar, 33Street J.C. Mahmood U. Ballon D. Alfieri A.A. Koutcher J.A. J. Biol. Chem. 1996; 271: 4113-4119Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 42Carmona A. Freedland R.A. Int. J. Biochem. 1990; 22: 595-599Crossref PubMed Scopus (10) Google Scholar, 43Kohler E. Barrach H.-J. Neubert D. FEBS Lett. 1970; 6: 225-228Crossref PubMed Scopus (118) Google Scholar, 44Street J.C. Alfieri A.A. Koutcher J.A. Cancer Res. 1997; 57: 3956-3962PubMed Google Scholar, 45Sudo J.-I. Ishihara A. Tanabe T. Jpn. J. Pharmacol. 1984; 36: 491-498Crossref PubMed Scopus (1) Google Scholar, 46Hashizume K. Onaya T. Sato A. Endocrinology. 1975; 97: 962-968Crossref PubMed Scopus (16) Google Scholar, 48Coper H. Neubert D. Biochim. Biophys. Acta. 1964; 89: 23-32PubMed Google Scholar). 6-Amino-NADP is an extremely potent competitive inhibitor of 6-PGDH, and concentrations that do not affect G6PDH may completely block 6-PGDH (43Kohler E. Barrach H.-J. Neubert D. FEBS Lett. 1970; 6: 225-228Crossref PubMed Scopus (118) Google Scholar). On the other hand, no inhibition of NAD-dependent enzymes results from the use of this compound (48Coper H. Neubert D. Biochim. Biophys. Acta. 1964; 89: 23-32PubMed Google Scholar). The inhibition of glycolysis from glucose by 6-AN makes it difficult to interpret the results using glucose as an energy source. Nevertheless, this compound is very valuable to study the effect of substrates such as fructose and glycerol that enter glycolysis beyond the formation of fructose 6-phosphate. When both the pentose phosphate pathway and phosphoglucoisomerase were inhibited with 6-AN and oxythiamine, stimulation of Glc-6-Pase gene expression by fructose or glycerol was not affected. These two substrates enter the glycolytic pathway at the level of triose phosphate(s), and the fact that the nonoxidative branch of the pentose phosphate pathway was blocked suggests that the pentose phosphate pathway is not required for the glucose effect on the Glc-6-Pase gene. In contrast to glucose, xylitol induced Glc-6-Pase mRNA even in the presence of inhibitors of the pentose phosphate pathway. Taken together, these results support the idea that xylitol and glucose signal through different pathways. The signaling pathway from glucose to the DNA sequences termed carbohydrate responsive element (ChoRE/GIRE) is not totally characterized. Phosphorylation/dephosphorylation of transcription factors have been implicated in the glucose responsiveness of many genes. Xylulose 5-phosphate, produced by the pentose phosphate pathway, has been suggested as a secondary messenger in sensing glucose concentration in the hepatocyte. This proposal stems from the fact that xylitol, a precursor of xylulose 5-phosphate, is able to stimulate the expression of a number of genes, both in vivo (12Massillon D. Chen W. Barzilai N. Prus-Wertheimer D. Hawkins M. Liu R. Taub R. Rossetti L. J. Biol. Chem. 1998; 273: 228-234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and in cultured cells (26Doiron B. Cuif M.-H. Chen R. Kahn A. J. Biol. Chem. 1996; 271: 5321-5324Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 28Mourrieras F. Foufelle F. Foretz M. Morin J. Bouche S. Ferre P. Biochem. J. 1997; 326: 345-349Crossref PubMed Scopus (68) Google Scholar). Xylulose 5-phosphate has been shown to activate the phosphatase 2A-mediated dephosphorylation (49Nishimura M. Uyeda K. J. Biol. Chem. 1995; 270: 26341-26346Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 50Liu Y.Q. Uyeda K. J. Biol. Chem. 1996; 271: 8824-8830Abstract Full Text PDF PubMed Scopus (31) Google Scholar) of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase and to decrease the activity of protein kinase A. The same phosphatase is also involved in the dephosphorylation of the transcription factor Sp1 (51Alberts A.S. Deng T. Lin A. Meinkoth J.L. Schonthal A. Mumby M.C. Karin M. Feramisco J.R. Mol. Cell. Biol. 1993; 13: 2104-2112Crossref PubMed Scopus (67) Google Scholar,52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Xylulose 5-phosphate-activated phosphatase is also activated by glucose or a glucose metabolite (50Liu Y.Q. Uyeda K. J. Biol. Chem. 1996; 271: 8824-8830Abstract Full Text PDF PubMed Scopus (31) Google Scholar). Furthermore, glucose-induced transcription of the acetyl-CoA carboxylase gene is mediated by the transcription factor Sp1 (52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). This stimulation is prevented by okadaic acid, an inhibitor of protein phosphatase types 1 and 2A (52Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). On the other hand, okadaic acid only partially inhibits the action of glucose and xylitol to stimulate the fructose-6-phosphate,2-kinase:fructose-2,6-biphosphatase (53Dupriez V.J. Rousseau G.G. Mol. Cell. Biol. 1997; 16: 1075-1085Google Scholar). These results suggest that either dephosphorylation of Sp1 is not universal in the glucose signaling pathway or that glucose signaling also involves other transcription factors such as the upstreamstimulatory factor proteins (54Lefrancois-Martinez A.-M Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: 2640-2643Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). These upstream stimulatory factor proteins were among the first transcription factors suggested to link glucose action to the ChoRE/GIRE (54Lefrancois-Martinez A.-M Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: 2640-2643Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 55Vaulont S. Kahn A. FASEB J. 1994; 8: 28-35Crossref PubMed Scopus (174) Google Scholar). The involvement of protein phosphatase(s) in the modulation of transcription factors involved in glucose signaling is worth exploring further. The metabolic consequences of increased Glc-6-Pase gene expression by glucose are not known. More than 25 years ago, Nordlie and colleagues (47Nordlie R.C. Hanson R.W. Mehlman M.A. Gluconeogenesis, Its Regulation in Mammalian Species. John Wiley & Sons, New York1976: 93-152Google Scholar) proposed the controversial idea that Glc-6-Pase, in the presence of high glucose levels, might act as a phosphotransferase that uses carbamoyl phosphate as the phosphate donor. Whether Glc-6-Pase plays any role in glucose phosphorylation is debatable. Nonetheless, the paradoxical induction of Glc-6-Pase gene by glucose points toward a potential role of this enzyme in the removal of glucose from the circulation. Glucose stimulation might also be needed to make sure that the enzyme does not disappear when the cell shifts from fed to starved conditions. One speculation is that glucose stimulation of Glc-6-Pase gene may take place in vivo when glucose cycling is needed following hyperglycemia. Glc-6-Pase is a very complex protein with different protein components that are not yet well characterized. I thank Dr. Richard W. Hanson for the use of his laboratory during the performance of much of this work. I also thank Frederic Bone for excellent assistance in various phases of this work and Dr. Ifeanyi J. Arinze for critical reading of the manuscript.

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