Glucose Catabolism Induces the L-Type Pyruvate Kinase Gene (125a) (original) (raw)
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Journal of Biological Chemistry, 1997
Twenty-six different hepatoma cell lines established from cancer-prone transgenic mice exhibited a close correlation between expression of the GLUT 2 glucose transporter and activation of the L-type pyruvate kinase (L-PK) gene by glucose, as judged by Northern blot analyses and transient transfection assays. The L-PK gene and a transfected L-PK construct were silent in GLUT 2(؉) cells and active in GLUT 2(؊) cells cultured in glucose-free medium. Transfection of GLUT 2(؊) cells with a GLUT 2 expression vector restored the inducibility of the L-PK promoter by glucose, mainly by suppressing the glucose-independent activity of this promoter. Culture of GLUT 2(؊) cells, in which the L-PK gene is constitutively expressed, in a culture medium using fructose as fuel selected GLUT 2(؉) clones in which the L-PK gene responded to glucose. The expression of the L-PK gene in GLUT 2(؊) cells cultured in the absence of glucose was correlated with a high intracellular glucose 6-phosphate (Glu-6-P) concentration while under similar culture conditions Glu-6-P concentration was very low in GLUT 2(؉) cells. Consequently, a role of GLUT 2 in the glucose responsiveness of glucose-sensitive genes in cultured hepatoma cells could be to allow for Glu-6-P depletion under gluconeogenic culture conditions. In the absence of GLUT 2, glucose endogeneously produced might be unable to be exported from the cells and would be phosphorylated again to Glu-6-P by constitutively expressed hexokinase isoforms, continuously generating the glycolytic intermediates active on the L-PK gene transcription.
Proceedings of the National Academy of Sciences, 2003
Carbohydrate-responsive element binding protein (ChREBP) is a transcription factor that activates lipogenic genes in liver in response to excess carbohydrate in the diet. ChREBP is regulated in a reciprocal manner by glucose and cAMP. cAMP-dependent protein kinase (protein kinase A) phosphorylates two physiologically important sites in ChREBP, Ser-196, which is located near nuclear localization signal sequence (NLS), and Thr-666, within the basic helix-loop-helix (bHLH) site, resulting in inactivation of nuclear translocation of ChREBP and of the DNA-binding activity, respectively. We demonstrate here that crude cytosolic extracts from livers of rats fed a high carbohydrate diet contained protein phosphatase (PPase) activity that dephosphorylated a peptide containing Ser-196, whereas a PPase in the nuclear extract catalyzed dephosphorylation of Ser-568 and Thr-666. All these PPases are activated specifically by xylulose 5-phosphate (Xu5P), but not by other sugar phosphates. Furthermore, addition of Xu5P elevated PPase activity to the level observed in extracts of fed liver cells. These partially purified PPases were characterized as PP2A-AB␦C by immunoblotting with specific antibodies. These results suggest that (ia) Xu5P-dependent PPase is responsible for activation of transcription of the L-type pyruvate kinase gene and lipogenic enzyme genes, and (ii) Xu5P is the glucose signaling compound. Thus, we propose that the same Xu5P-activated PPase controls both acute and longterm regulation of glucose metabolism and fat synthesis.
The immunological and catalytically active form of L-type pyruvate kinase in rat liver cytosol
The International journal of biochemistry, 1983
The protein species precipitated from rat liver cytosol by rabbit antisera raised to pure L-type pyruvate kinase were investigated by sodium dodecyl sulphate gel electrophoresis. The primary antisera (anti-L-type pyruvate kinase) precipitated protein species with mol. wts 56,000, 41,000 and 39,000. The 41,000 mol. wt protein was identified as fructose-bis-phosphatase. Double diffusion and immunotitration experiments established that L-type pyruvate kinase and fructose-bis-phosphatase shared common antigenic determinants. This information enabled an improved antiserum (anti-LPK) to be obtained. The use of anti-LPK showed that the 56,000 mol. wt subunit was the only catalytically and immunologically active form of L-type pyruvate kinase in liver. This was confirmed by biosynthetic experiments with cultured hepatocytes. The specific activity of the enzyme in liver extracts was also determined by quantitative immunotitration with anti-LPK. Despite changes in dietary status which varied ...
Journal of Biological Chemistry, 1999
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 (4fold) 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.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1995
Glycogen synthesis in hepatocytes was determined at various concentrations of CO 2 and medium HCO 3 to modulate cell pH. Glycogen synthesis from glucose was highest in acidic conditions (5% CO2/12.5 mM HCO 3) and lowest in alkaline conditions (2.5% CO2/25 mM HCO~-). Physiological concentrations of lactate/pyruvate (2 mM/0.2 mM) stimulated the conversion of glucose to glycogen in all media examined and mercaptopicolinate, an inhibitor of gluconeogenesis, caused a similar stimulation as lactate/pyruvate. In alkaline media, the s~Eimulation by mercaptopicolinate and by lactate/pyruvate was additive, indicating that the latter is not due to gluconeogenic flux. In acidic media, the stimulation by both lactate/pyruvate and mercaptopicolinate was inhibited by amiloride, an inhibitor of Na+/H + exchange. Since Na+/H ÷ exchange is activated when cell pH falls below a certain threshold, it is postulated that lactate and pyruvate stimulate the conversion of glucose to glycogen through cellular acidification.
Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes
European journal of biochemistry / FEBS, 1990
Fructose 1-phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1-phosphate and fructose. The concentration of fructose 1-phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05-1 mM) caused a concentration-dependent and transient increase in the fructose 1-phosphate content. Glucagon (1 microM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1-phosphate in cells incubated with fructose, whereas the addition of 0.1 microM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1-phosphate concentration. Sorbitol had a similar effect to fructose in causing the formation of fructose 1-phosphate. D...
Journal of Biological Chemistry, 1999
Transcription of the liver type pyruvate kinase and lipogenesis enzyme genes is induced by high carbohydrate in liver. We have found a novel protein factor in rat liver nuclei that binds to the glucose response element (CACGTG motifs) of the pyruvate kinase gene (Liu, Z., Thompson, K. S., and Towle, H. C. (1993) J. Biol. Chem. 268,12787-12795) and the "insulin response element" of fatty acid synthase gene. The amounts of this DNA-binding protein, termed "glucose response element binding protein" (GRBP) in the nuclear extract, were increased in liver by a high carbohydrate diet and decreased by starvation, high fat, and high protein diet. GRBP also occurs in cytosols of liver and is dependent on carbohydrate. Both the nuclear and the cytosolic GRBP showed similar properties, except the former was more resistant to thermal inactivation than the latter. Kinetics of glucose activation of the cytosolic GRBP in a primary culture of hepatocytes indicated that a half-maximum activation was achieved after 6 h, and glucose concentration required for the maximum activation of the GRBP was approximately 12 mM. Dibutyryl-cAMP, okadaic acid, and forskolin inhibited glucose activation of both GRBP and liver pyruvate kinase transcription. These results suggested that GRBP may be a factor that recognizes the glucose response motif site and may be involved in mediating carbohydrate response of the pyruvate kinase gene.