Glucose-6-phosphatase Overexpression Lowers Glucose 6-Phosphate and Inhibits Glycogen Synthesis and Glycolysis in Hepatocytes without Affecting Glucokinase Translocation
1999; Elsevier BV; Volume: 274; Issue: 35 Linguagem: Inglês
10.1074/jbc.274.35.24559
ISSN1083-351X
AutoresSusan Aiston, Khiet Y. Trinh, Alex J. Lange, Christopher B. Newgard, Loranne Agius,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoIn hepatocytes glucokinase (GK) and glucose-6-phosphatase (Glc-6-Pase)1 have converse effects on glucose 6-phosphate (and fructose 6-phosphate) levels. To establish whether hexose 6-phosphate regulates GK binding to its regulatory protein, we determined the effects of Glc-6-Pase overexpression on glucose metabolism and GK compartmentation. Glc-6-Pase overexpression (4-fold) decreased glucose 6-phosphate levels by 50% and inhibited glycogen synthesis and glycolysis with a greater negative control coefficient on glycogen synthesis than on glycolysis, but it did not affect the response coefficients of glycogen synthesis or glycolysis to glucose, and it did not increase the control coefficient of GK or cause dissociation of GK from its regulatory protein, indicating that in hepatocytes fructose 6-phosphate does not regulate GK translocation by feedback inhibition. GK overexpression increases glycolysis and glycogen synthesis with a greater control coefficient on glycogen synthesis than on glycolysis. On the basis of the similar relative control coefficients of GK and Glc-6-Pase on glycogen synthesis compared with glycolysis, and the lack of effect of Glc-6-Pase overexpression on GK translocation or the control coefficient of GK, it is concluded that the main regulatory function of Glc-6-Pase is to buffer the glucose 6-phosphate concentration. This is consistent with recent findings that hyperglycemia stimulates Glc-6-Pase gene transcription. In hepatocytes glucokinase (GK) and glucose-6-phosphatase (Glc-6-Pase)1 have converse effects on glucose 6-phosphate (and fructose 6-phosphate) levels. To establish whether hexose 6-phosphate regulates GK binding to its regulatory protein, we determined the effects of Glc-6-Pase overexpression on glucose metabolism and GK compartmentation. Glc-6-Pase overexpression (4-fold) decreased glucose 6-phosphate levels by 50% and inhibited glycogen synthesis and glycolysis with a greater negative control coefficient on glycogen synthesis than on glycolysis, but it did not affect the response coefficients of glycogen synthesis or glycolysis to glucose, and it did not increase the control coefficient of GK or cause dissociation of GK from its regulatory protein, indicating that in hepatocytes fructose 6-phosphate does not regulate GK translocation by feedback inhibition. GK overexpression increases glycolysis and glycogen synthesis with a greater control coefficient on glycogen synthesis than on glycolysis. On the basis of the similar relative control coefficients of GK and Glc-6-Pase on glycogen synthesis compared with glycolysis, and the lack of effect of Glc-6-Pase overexpression on GK translocation or the control coefficient of GK, it is concluded that the main regulatory function of Glc-6-Pase is to buffer the glucose 6-phosphate concentration. This is consistent with recent findings that hyperglycemia stimulates Glc-6-Pase gene transcription. glucose-6-phosphatase The relative activities of hepatic glucokinase and glucose-6-phosphatase, which catalyze the first and last steps in glucose utilization and production, respectively, are thought to have a major role in regulating blood glucose homeostasis (1Barzilai N. Rossetti L. J. Biol. Chem. 1993; 268: 25019-25025Abstract Full Text PDF PubMed Google Scholar, 2Mevorach M. Giacca A. Aharon Y. Hawkins M. Shamoon H. Rossetti L. J. Clin. Invest. 1998; 102: 744-753Crossref PubMed Scopus (128) Google Scholar, 3Argaud D. Zhang Q. Pan W. Maitra S. Pilkis S.J. Lange A.J. Diabetes. 1996; 45: 1563-1571Crossref PubMed Google Scholar). The activities of these two enzymes change in a converse manner during fasting and refeeding or during insulin deficiency and insulin treatment. Fasting and insulin deficiency are associated with inhibition of glucokinase transcription and with a gradual decline in total glucokinase activity, whereas refeeding or insulin treatment restores glucokinase activity by induction of glucokinase transcription (4Iyndejian P.B. Biochem. J. 1993; 293: 1-13Crossref PubMed Scopus (278) Google Scholar, 5Iynedjian P.B. Jotterand D. Nouspikel T. Asfari M. Pilot P.R. J. Biol. Chem. 1989; 264: 21824-21829Abstract Full Text PDF PubMed Google Scholar). Conversely, the transcription of glucose-6-phosphatase is negatively regulated by insulin, and the activity of glucose-6-phosphatase is markedly increased in fasted or insulin-deficient diabetic states (6Lange 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, 7Streeper R.S. Svitek C.A. Chapman S. Greenbaum L.E. Taub T. O'Brien R.M. J. Biol. Chem. 1997; 272: 11698-11701Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In addition to changes in total enzyme concentration by regulation of gene transcription, glucokinase activity is also regulated acutely by a translocation mechanism (8Agius L. Peak M. Biochem. J. 1993; 296: 785-796Crossref PubMed Scopus (132) Google Scholar, 9Agius L. Peak M. Van Schaftingen E Biochem. J. 1995; 309: 711-713Crossref PubMed Scopus (64) Google Scholar, 10Brown K.S. Kalinowski S.S. Megill J.R. Durham S.K. Mookhtiar K.A. Diabetes. 1997; 46: 179-186Crossref PubMed Scopus (115) Google Scholar). This involves the sequestration of glucokinase in an inactive state in the nucleus (10Brown K.S. Kalinowski S.S. Megill J.R. Durham S.K. Mookhtiar K.A. Diabetes. 1997; 46: 179-186Crossref PubMed Scopus (115) Google Scholar) bound to a 68-kDa regulatory protein at low concentrations of extracellular glucose (8Agius L. Peak M. Biochem. J. 1993; 296: 785-796Crossref PubMed Scopus (132) Google Scholar, 9Agius L. Peak M. Van Schaftingen E Biochem. J. 1995; 309: 711-713Crossref PubMed Scopus (64) Google Scholar). A rise in extracellular glucose or low concentrations of fructose or sorbitol cause the rapid dissociation of glucokinase from its regulatory protein and the translocation of the enzyme to the cytoplasm. This translocation mechanism results in a large increase in glucokinase activity in the cytoplasm within minutes of a rise in extracellular glucose concentration (8Agius L. Peak M. Biochem. J. 1993; 296: 785-796Crossref PubMed Scopus (132) Google Scholar, 10Brown K.S. Kalinowski S.S. Megill J.R. Durham S.K. Mookhtiar K.A. Diabetes. 1997; 46: 179-186Crossref PubMed Scopus (115) Google Scholar). The binding properties of glucokinase to its 68-kDa regulatory protein have been extensively characterized from studies on the purified proteins (11Van Schaftingen E. Eur. J. Biochem. 1989; 179: 179-184Crossref PubMed Scopus (160) Google Scholar, 12Vandercammen A. Van Schaftingen E. Eur. J. Biochem. 1990; 191: 483-489Crossref PubMed Scopus (99) Google Scholar, 13Vandercammen A. Van Schaftingen E. Eur. J. Biochem. 1991; 200: 545-551Crossref PubMed Scopus (81) Google Scholar, 14Detheux M. Vandercammen A. Van Schaftingen E. Eur. J. Biochem. 1991; 200: 553-561Crossref PubMed Scopus (57) Google Scholar, 15Van Schaftingen E. Veiga-da-Cunha M. Niculescu L. Biochem. Soc. Trans. 1997; 25: 136-140Crossref PubMed Scopus (61) Google Scholar). Binding of glucokinase to the regulatory protein is enhanced by fructose 6-phosphate, and this effect is antagonized by fructose 1-phosphate. Both ligands bind to the same site on the regulatory protein and alter its affinity for glucokinase. It has been proposed that the rise in fructose 6-phosphate in hepatocytes during active glycogenolysis and gluconeogenesis causes increased binding of glucokinase to the regulatory protein and decreased glucose phosphorylation (15Van Schaftingen E. Veiga-da-Cunha M. Niculescu L. Biochem. Soc. Trans. 1997; 25: 136-140Crossref PubMed Scopus (61) Google Scholar, 16Van Schaftingen E. Biochem. J. 1995; 308: 23-29Crossref PubMed Scopus (30) Google Scholar, 17Niculescu L. Van Schaftingen E. Diabetologia. 1998; 41: 947-954Crossref PubMed Scopus (8) Google Scholar), whereas the increase in fructose 1-phosphate that results from metabolism of fructose or sorbitol causes dissociation of glucokinase from the regulatory protein (9Agius L. Peak M. Van Schaftingen E Biochem. J. 1995; 309: 711-713Crossref PubMed Scopus (64) Google Scholar, 18Niculescu L. Veiga-da-Cunha M. Van Schaftingen E. Biochem. J. 1997; 321: 239-246Crossref PubMed Scopus (46) Google Scholar). Although the role of fructose 1-phosphate in explaining the translocation of glucokinase by fructose and sorbitol is now firmly established (18Niculescu L. Veiga-da-Cunha M. Van Schaftingen E. Biochem. J. 1997; 321: 239-246Crossref PubMed Scopus (46) Google Scholar), the role of changes in fructose 6-phosphate in regulating the binding of glucokinase to its regulatory protein in the intact cell remains a contentious issue, with arguments both for (15Van Schaftingen E. Veiga-da-Cunha M. Niculescu L. Biochem. Soc. Trans. 1997; 25: 136-140Crossref PubMed Scopus (61) Google Scholar, 16Van Schaftingen E. Biochem. J. 1995; 308: 23-29Crossref PubMed Scopus (30) Google Scholar, 17Niculescu L. Van Schaftingen E. Diabetologia. 1998; 41: 947-954Crossref PubMed Scopus (8) Google Scholar) and against (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar) a physiological role for changes in fructose 6-phosphate in the intact cell. Adenovirus-mediated glucose-6-phosphatase overexpression markedly suppresses the hepatocyte glucose 6-phosphate content (20Seoane J. Trinh K. O'Doherty R.M. Gomez-Foix A.M. Lange A.J. Newgard C.B. Guinovart J.J. J. Biol. Chem. 1997; 272: 26972-26977Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and thereby fructose 6-phosphate which is in equilibrium with glucose 6-phosphate (16Van Schaftingen E. Biochem. J. 1995; 308: 23-29Crossref PubMed Scopus (30) Google Scholar) and thus provides a powerful tool to unequivocally test the physiological role of changes in hexose 6-phosphate in the intact cell.In order to evaluate the regulatory function of glucose-6-phosphatase in the hepatocyte, this study had three aims. The first was to determine the role of changes in hepatocyte hexose 6-phosphate content in regulating glucokinase translocation. The second was to determine the control strengths (control coefficients) of glucose-6-phosphatase and glucokinase on glycolysis and glycogen synthesis. The third was to determine whether glucose-6-phosphatase overexpression alters the control strength of glucokinase on glycolysis or glycogen synthesis or the response coefficients of these pathways to glucose in the hepatocyte. The results support a hypothesis that the primary regulatory function of glucose-6-phosphatase in the hepatocyte is to buffer the glucose 6-phosphate concentration. This hypothesis is consistent with recent apparently paradoxical findings on glucose-6-phosphatase activity in experimental and pathological states.DISCUSSIONGlucokinase (hexokinase IV) differs from the other hexokinase isoenzymes (I–III) in that it is not inhibited by physiological concentrations of glucose 6-phosphate, the product of the reaction (4Iyndejian P.B. Biochem. J. 1993; 293: 1-13Crossref PubMed Scopus (278) Google Scholar). Since binding of purified glucokinase to the regulatory (inhibitory) protein is enhanced by fructose 6-phosphate (11Van Schaftingen E. Eur. J. Biochem. 1989; 179: 179-184Crossref PubMed Scopus (160) Google Scholar, 13Vandercammen A. Van Schaftingen E. Eur. J. Biochem. 1991; 200: 545-551Crossref PubMed Scopus (81) Google Scholar), it has been proposed that fructose 6-phosphate is a substitute for end product inhibition by glucose 6-phosphate because fructose 6-phosphate and glucose 6-phosphate are maintained in equilibrium by phosphoglucoisomerase (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar). This hypothesis was tested using mannitol which is metabolized by hepatocytes to mannitol 1-phosphate (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar). Mannitol was shown to inhibit glucose metabolism and glucokinase translocation. Since mannitol 1-phosphate can act as an analogue of fructose 6-phosphate in promoting binding of glucokinase to the regulatory protein (14Detheux M. Vandercammen A. Van Schaftingen E. Eur. J. Biochem. 1991; 200: 553-561Crossref PubMed Scopus (57) Google Scholar), these findings appeared to support the hypothesis that binding of glucokinase to the regulatory protein in intact hepatocytes is regulated by analogues of fructose 6-phosphate (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar). However, the possibility that the inhibitory effect of mannitol on detritiation of glucose was due to other mechanisms could not be rigorously excluded since mannitol 1-phosphate is a potent inhibitor of phosphoglucoisomerase (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar). Direct evidence in support of regulation of glucokinase translocation in intact hepatocytes by changes in fructose 6-phosphate/glucose 6-phosphate is still lacking.The first aim of this study was to determine whether lowering the hexose 6-phosphate content of hepatocytes by glucose-6-phosphatase overexpression causes dissociation of glucokinase from its regulatory protein as assessed from the distribution of glucokinase between free and bound states and from the response coefficients of glycogen synthesis and glycolysis to glucose. If changes in the hepatocyte hexose 6-phosphate content have a physiological role in regulating the binding of glucokinase to its regulatory protein, then lowering of the hexose 6-phosphate content with glucose-6-phosphatase would be expected to potentiate the translocation of glucokinase in response to glucose or sorbitol and to increase the response coefficient of glycolysis and glycogen synthesis to glucose. Our results demonstrate that lowering of the hepatocyte hexose 6-phosphate content by glucose-6-phosphatase overexpression does not affect glucokinase translocation at any concentration of glucose (5–35 mm) or sorbitol, as determined from the free glucokinase activity that is a measure of the cytoplasmic enzyme (9Agius L. Peak M. Van Schaftingen E Biochem. J. 1995; 309: 711-713Crossref PubMed Scopus (64) Google Scholar, 10Brown K.S. Kalinowski S.S. Megill J.R. Durham S.K. Mookhtiar K.A. Diabetes. 1997; 46: 179-186Crossref PubMed Scopus (115) Google Scholar). Consistent with the unchanged cytoplasmic glucokinase activity, no increase in the response coefficients of glycogen synthesis or glycolysis to glucose was noted. The glucose 6-phosphate content of hepatocytes increases more than 2-fold between 5 and 25 mm glucose (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar, 20Seoane J. Trinh K. O'Doherty R.M. Gomez-Foix A.M. Lange A.J. Newgard C.B. Guinovart J.J. J. Biol. Chem. 1997; 272: 26972-26977Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and it is markedly decreased by glucose-6-phosphatase overexpression (20Seoane J. Trinh K. O'Doherty R.M. Gomez-Foix A.M. Lange A.J. Newgard C.B. Guinovart J.J. J. Biol. Chem. 1997; 272: 26972-26977Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The translocation of glucokinase by increasing glucose concentration (despite the increase in glucose 6-phosphate/fructose 6-phosphate levels) is explained by the effect of glucose on dissociation of glucokinase from the regulatory protein (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar). The present finding that lowering the hexose 6-phosphate concentration by glucose-6-phosphatase overexpression (over a range of substrate concentrations) does not cause dissociation of glucokinase from its regulatory protein indicates that physiological changes in hexose 6-phosphate do not regulate glucokinase binding to the regulatory protein by feedback inhibition. It seems likely, therefore, that the regulatory protein is saturated with fructose 6-phosphate under most conditions. These findings support the conclusion that the two main functions of the regulatory protein are for feed-forward activation of glucokinase by glucose and for stimulation of glucose metabolism by precursors of fructose 1-phosphate but not for feedback inhibition by glucose 6-phosphate/fructose 6-phosphate. A major function of the liver is the maintenance of blood glucose homeostasis. Regulation of glucokinase translocation by feed-forward activation by glucose without feedback inhibition by hexose 6-phosphate is consistent with such a function.The second aim of this study was to gain insight into the regulatory function of glucose-6-phosphatase in hepatocytes. The recognition that the rate of glucose phosphorylation in hepatocytes, estimated from the detritiation of [2-3H]glucose, exceeds the rate of glucose metabolism has led to several hypotheses on the possible regulatory function(s) of the glucose/glucose 6-phosphate cycle (32Hue L. Adv. Enzymol. Relat. Areas Mol. Biol. 1981; 52: 247-331PubMed Google Scholar,33Clark D.G. Rognstad R. Katz J. Biochem. Biophys. Res. Commun. 1973; 54: 1141-1148Crossref PubMed Scopus (54) Google Scholar). Suggested functions of substrate cycles in general include the following: increased sensitivity of regulation; control of the direction of flux at metabolic branchpoints; buffering of metabolite concentrations; balancing ATP production and utilization by regenerating ADP and thermogenesis (31Fell D. Snell K. Understanding the Control of Metabolism. Portland Press Ltd., London1997: 219-225Google Scholar, 33Clark D.G. Rognstad R. Katz J. Biochem. Biophys. Res. Commun. 1973; 54: 1141-1148Crossref PubMed Scopus (54) Google Scholar). To investigate the regulatory function of glucose-6-phosphatase in the hepatocyte, we used metabolic control analysis to determine the control strength of glucose-6-phosphatase on glycogen synthesis and glycolysis, and we determined whether glucose-6-phosphatase overexpression alters the control strength of glucokinase. Metabolic control analysis is a very useful analytical approach to probe the regulatory mechanisms that operate in the intact cell. Kacser and Burns (29Kacser H. Burns J.A. Biochem. Soc. Trans. 1979; 7: 1149-1160Crossref PubMed Scopus (322) Google Scholar) showed that the rate of flux through a metabolic pathway approximates a hyperbolic function of the activity (or concentration) of constituent enzymes of the pathway. The control coefficient (control strength) of an enzyme is a measure of the sensitivity of pathway flux to changes in the concentration (activity) of the enzyme and can be determined either from the slope of the curve of pathway flux against enzyme concentration multiplied by the scaling factor or from the slope of a double logarithmic plot of flux against enzyme concentration (29Kacser H. Burns J.A. Biochem. Soc. Trans. 1979; 7: 1149-1160Crossref PubMed Scopus (322) Google Scholar, 30Fell D.A. Biochem. J. 1992; 286: 313-330Crossref PubMed Scopus (632) Google Scholar). Since the relation between flux and enzyme concentration is hyperbolic, the control coefficient of enzymes generally decreases with increasing enzyme concentration. We showed previously (21Agius L. Peak M. Newgard C.B. Gomez-Foiz A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) that glucokinase has a very high control strength on glycogen synthesis, and this is dependent on the glucose concentration. We proposed that the translocation of glucokinase between the nucleus and the cytoplasm is a major contributing factor to the high control strength and accounts for the glucose dependence (28Agius L. Adv. Enzyme Regul. 1998; 38: 303-331Crossref PubMed Scopus (61) Google Scholar). At low glucose concentration when glucokinase is sequestered in an inactive state in the nucleus and the activity of cytoplasmic activity is minimal, a small increase in total glucokinase activity by adenovirus-mediated overexpression is associated with a larger fractional increase in cytoplasmic activity (compared with total activity) and accordingly with a large fractional increase in glycogen synthesis (21Agius L. Peak M. Newgard C.B. Gomez-Foiz A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 28Agius L. Adv. Enzyme Regul. 1998; 38: 303-331Crossref PubMed Scopus (61) Google Scholar). At higher glucose concentrations when glucokinase is equally distributed between the nucleus and the cytoplasm (or present predominantly in the cytoplasm), an increase in total enzyme activity by overexpression is associated with a similar fractional increase in cytoplasmic activity and, accordingly, with a lower fractional stimulation of glycogen synthesis than at low glucose concentration (28Agius L. Adv. Enzyme Regul. 1998; 38: 303-331Crossref PubMed Scopus (61) Google Scholar).The present results show first that glucose-6-phosphatase overexpression inhibits glycogen synthesis and glycolysis from glucose without affecting glucokinase translocation. Second, they show that both glucokinase and glucose-6-phosphatase have a greater control coefficient (positive and negative, respectively) on glycogen synthesis than on glycolysis. Third, they show that the control coefficient of glucokinase but not that of glucose-6-phosphatase is dependent on glucose concentration. Fourth, they show that the control coefficient of glucokinase on glycogen synthesis is greater than the control coefficient of glucose-6-phosphatase. Finally, glucose-6-phosphatase overexpression does not increase the control coefficient of glucokinase. The latter finding does not support a regulatory role for glucose-6-phosphatase in increasing the sensitivity of regulation of glucose metabolism. The findings that the control coefficient of glucokinase but not that of glucose-6-phosphatase is dependent on glucose concentration and that glucokinase has a greater control coefficient than glucose-6-phosphatase, particularly at low glucose, are consistent with our previous hypothesis that the compartmentation of glucokinase in the hepatocyte is a major contributing factor to both the high control strength at low glucose concentration and to the glucose dependence of the control strength (21Agius L. Peak M. Newgard C.B. Gomez-Foiz A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 28Agius L. Adv. Enzyme Regul. 1998; 38: 303-331Crossref PubMed Scopus (61) Google Scholar).Glucokinase and glucose-6-phosphatase have converse effects on the hepatocyte glucose 6-phosphate content (20Seoane J. Trinh K. O'Doherty R.M. Gomez-Foix A.M. Lange A.J. Newgard C.B. Guinovart J.J. J. Biol. Chem. 1997; 272: 26972-26977Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 34Seoane J. Gomez-Foix A.M. O'Doherty R.M. Gomez-Ara C. Newgard C.B. Guinovart J.J. J. Biol. Chem. 1996; 271: 23756-23760Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The present study shows converse effects on glycolysis and glycogen synthesis with both glucokinase and glucose-6-phosphatase having a greater control strength on glycogen synthesis than on glycolysis. Based on these findings that overexpression of glucose-6-phosphatase has no effect on glucokinase translocation and that it also does not increase the control strength of glucokinase, we propose that the main regulatory function of glucose-6-phosphatase is to buffer the glucose 6-phosphate concentration in the hepatocyte. It is noteworthy that in the intact cell the buffering role of the glucose-6-phosphatase system would be determined not only by changes in expression of the catalytic subunit of glucose-6-phosphatase but also by the glucose 6-phosphate transporter which determines the kinetics of entry of glucose 6-phosphate into the endoplasmic reticulum.The role of glucose 6-phosphate in regulating both glycolysis and glycogen synthesis in the hepatocyte is well established. Glucose 6-phosphate regulates glycolysis by increasing the concentration of fructose 2,6-bisphosphate, a potent allosteric activator of phosphofructokinase-1 (35Hers H.-G. Van Schaftingen E. Biochem. J. 1982; 206: 1-12Crossref PubMed Scopus (308) Google Scholar, 36Hue L. Rider M.H. Biochem. J. 1987; 245: 313-324Crossref PubMed Scopus (332) Google Scholar). Substrate-induced translocation of glucokinase is associated with a marked increase in glucose 6-phosphate and fructose 2,6-bisphosphate in hepatocytes, and the latter explains the stimulation of glycolysis by sorbitol (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar, 28Agius L. Adv. Enzyme Regul. 1998; 38: 303-331Crossref PubMed Scopus (61) Google Scholar). Glucose 6-phosphate increases glycogen synthesis both by acting as an allosteric activator of glycogen synthase and by increasing the dephosphorylation state of glycogen synthase by rendering the enzyme a better substrate for synthase phosphatase (37Villar-Palasi C. Guinovart J.J. FASEB J. 1997; 11: 544-558Crossref PubMed Scopus (158) Google Scholar). The latter effect has been established from studies demonstrating a correlation between the activation state of glycogen synthase and glucose 6-phosphate in hepatocytes overexpressing glucokinase (34Seoane J. Gomez-Foix A.M. O'Doherty R.M. Gomez-Ara C. Newgard C.B. Guinovart J.J. J. Biol. Chem. 1996; 271: 23756-23760Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar).The present hypothesis that the primary regulatory function of glucose-6-phosphatase is to buffer the concentration of glucose 6-phosphate, a pivotal regulator of glycogen synthesis and glycolysis, is consistent with various recent apparently paradoxical findings on glucose-6-phosphatase. First, studies both in vivo (38Massillon 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) and in vitro on isolated hepatocytes or hepatoma cell lines (6Lange 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,39Argaud D. Kirby T.L. Newgard C.B. Lange A.J. J. Biol. Chem. 1997; 272: 12854-12861Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 40Chatelain F. Pegorier J.P. Minassian C. Bruni N. Tarpin S. Girard J. Mithieux G. Diabetes. 1998; 47: 882-889Crossref PubMed Scopus (78) Google Scholar) have shown that high glucose concentrations induce glucose-6-phosphatase gene transcription. If the primary regulatory role of glucose-6-phosphatase in the liver cell were to control gluconeogenesis then induction of transcription by glucose is unexpected. Accordingly, the transcription of hepatic phosphoenolpyruvate carboxykinase, which unlike glucose-6-phosphatase has an exclusive role in gluconeogenesis, is repressed by glucose metabolism (41Scott D.K. O'Doherty R.M. Stafford J.M. Newgard C.B. Granner D.K. J. Biol. Chem. 1998; 273: 24145-24151Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, if the primary regulatory function of glucose-6-phosphatase were to buffer the glucose 6-phosphate concentration then induction by hyperglycemia is not a pathological consequence of uncontrolled diabetes (38Massillon 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) but a compensatory mechanism. We have shown recently that glucose-6-phosphatase overexpression in vivo increases blood glucose levels in the fed state and when fasted rats are challenged with an oral glucose tolerance test but paradoxically not in the fasted state (42Trinh K.Y. O'Doherty R.M. Anderson P. Lange A.J. Newgard C.B. J. Biol. Chem. 1998; 273: 31615-31620Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). This finding can also be explained by the primary regulatory role of glucose-6-phosphatase in buffering glucose 6-phosphate levels in the absorptive state when glucokinase is in the cytoplasmic compartment. Glycogenolysis and gluconeogenesis are the two sources of glucose 6-phosphate for hepatic glucose production during fasting. The rate of glycogenolysis is determined by the activity of phosphorylase. The regulation of gluconeogenesis has been extensively studied by Groen and co-workers (43Groen A.K. van Roermund C.W.T. Vervoorn R.C. Tager J.M. Biochem. J. 1986; 237: 379-389Crossref PubMed Scopus (145) Google Scholar) who determined the control coefficients of the gluconeogenic enzymes in hepatocytes from starved rats and demonstrated the importance of regulation at pyruvate kinase and pyruvate carboxylase. The flux control coefficient of glucose-6-phosphatase on gluconeogenesis was found to be remarkably low (∼0.02), and the glucose 6-phosphate concentration was far below the Km (43Groen A.K. van Roermund C.W.T. Vervoorn R.C. Tager J.M. Biochem. J. 1986; 237: 379-389Crossref PubMed Scopus (145) Google Scholar). Accordingly the “flux-generating steps” of hepatic glucose production are phosphorylase and pyruvate metabolism at the phosphoenolpyruvate branchpoint. This would explain why glucose-6-phosphatase overexpression does not elevate fasting glycemia (42Trinh K.Y. O'Doherty R.M. Anderson P. Lange A.J. Newgard C.B. J. Biol. Chem. 1998; 273: 31615-31620Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). It does, however, markedly elevate glucose levels in the fed state and particularly the response to an oral glucose tolerance test (42Trinh K.Y. O'Doherty R.M. Anderson P. Lange A.J. Newgard C.B. J. Biol. Chem. 1998; 273: 31615-31620Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), consistent with the present hypothesis of a role of glucose-6-phosphatase in buffering the glucose 6-pho
Referência(s)