An Unusual High-Km Hexokinase Is Expressed in the mhAT3F Hepatoma Cell Line (original) (raw)
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Journal of Biological Chemistry, 1996
Glucokinase reversibly partitions between a bound and a free state in the hepatocyte in response to the metabolic status of the cell. Maximum binding occurs at low [glucose] (<5 mM) and minimum binding at high [glucose] or in the presence of sorbitol or fructose. In this study we determined the binding characteristics of glucokinase in the hepatocyte in situ, by adenovirusmediated glucokinase overexpression combined with the digitonin-permeabilization technique. We also determined the sensitivity of glycogen synthesis to changes in either total glucokinase overexpression or in free glucokinase activity. Glucokinase overexpression is associated with an increase in both free and bound activity, with an overall decrease in the proportion of bound activity. In hepatocytes incubated at low [glucose] (0 -5 mM), glucokinase binding involves a high-affinity binding site with a K d of ϳ0.1 M and a binding capacity of ϳ3 pmol/mg total cell protein and low-affinity binding with a K d of ϳ1.6 M. Increasing glucose concentration to 20 mM causes a dose-dependent increase in the K d of the high-affinity site to ϳ0.6 M, and this effect was mimicked by 50 M sorbitol, a precursor of fructose 1-P, confirming that this site is the regulatory protein of glucokinase.
Journal of Biological Chemistry, 2002
Using adenovirus-mediated gene transfer into FTO-2B cells, a rat hepatoma cell line, we have overexpressed hexokinase I (HK I), glucokinase (GK), liver glycogen synthase (LGS), muscle glycogen synthase (MGS), and combinations of each of the two glucose-phosphorylating enzymes with each one of the GS isoforms. FTO-2B cells do not synthesize glycogen even when incubated with high doses of glucose. Adenovirus-induced overexpression of HK I and/or LGS, two enzymes endogenously expressed by these cells, did not produce a significant increase in the levels of active GS and the total glycogen content. In contrast, GK overexpression led to the glucosedependent activation of endogenous or overexpressed LGS and to the accumulation of glycogen. Similarly overexpressed MGS was efficiently activated by the glucose-6-phosphate (Glc-6-P) produced by either endogenous or overexpressed HK I and by overexpressed GK. These results indicate the existence of at least two pools of Glc-6-P in the cell, one of them is accessible to both isoforms of GS and is replenished by the action of GK, whereas LGS is excluded from the cellular compartment where the Glc-6-P produced by HK I is directed. These findings are interpreted in terms of the metabolic role that the two pairs of enzymes, HK I-MGS in the muscle and GK-LGS in the hepatocyte, perform in their respective tissues.
Tissue-Specific regulation of glucokinase gene expression
Journal of Cellular Biochemistry, 1992
Glucokinase contributes to the maintenance of blood glucose homeostasis by catalyzing the high K, phosphorylation of glucose in the liver and the pancreatic P cell, the only two tissues known to express this enzyme. Molecular biological studies of the glucokinase gene and its products have advanced our understanding of how this gene is differentially regulated in the liver and P cell. The production of an active glucokinase isoform is determined by both transcriptional and post-transcriptional events. Two different promoter regions that are widely separated in a single glucokinase gene are used to produce glucokinase mRNAs in the liver, pancreatic p cell, and pituitary. The different transcription control regions are tissue-specific in their expression and are differentially regulated. In liver, glucokinase gene expression is regulated by insulin and CAMP, whereas in the cell it is regulated by glucose. The upstream glucokinase promoter region, which gives rise to the glucokinase mRNA in pituitary and pancreas, is structurally and functionally different from the downstream promoter region, which gives rise to the glucokinase mRNA in liver. The use of distinct promoter regions in a single glucokinase gene enables a different set of transcription factors to be utilized in the liver and islet, thus allowing a functionally similar gene product to be regulated in a manner consistent with the different functions of these two tissues. In addition, the splicing of the glucokinase pre-mRNA is regulated in a tissue-specific manner and can affect the activity of the gene product. This is most apparent in the pituitary where an alternately spliced glucokinase mRNA is produced that does not encode a functional enzyme due to an introduced frameshift.
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.
Differential Effects of Overexpressed Glucokinase and Hexokinase I in Isolated Islets
Journal of Biological Chemistry, 1996
Glucose-stimulated insulin secretion is believed to require metabolism of the sugar via a high K m pathway in which glucokinase (hexokinase IV) is rate-limiting. In this study, we have used recombinant adenoviruses to overexpress the liver and islet isoforms of glucokinase as well as low K m hexokinase I in isolated rat islets of Langerhans. Glucose phosphorylating activity increased by up to 20-fold in extracts from islets treated with adenoviruses containing the cDNAs encoding either tissue isoform of glucokinase, but such cells exhibited no increase in 2-or 5-[ 3 H]glucose usage, lactate production, glycogen content, or glucose oxidation. Furthermore, glucokinase overexpression enhanced insulin secretion in response to stimulatory glucose or glucose plus arginine by only 36 -53% relative to control islets. In contrast to the minimal effects of overexpressed glucokinases, overexpression of hexokinase I caused a 2.5-4-fold enhancement in all metabolic parameters except glycogen content when measured at a basal glucose concentration (3 mM). Based on measurement of glucose phosphorylation in intact cells, overexpressed glucokinase is clearly active in a non-islet cell line (CV-1) but not within islet cells. That this result cannot be ascribed to the levels of glucokinase regulatory protein in islets is shown by direct measurement of its activity and mRNA. These data provide evidence for functional partitioning of glucokinase and hexokinase and suggest that overexpressed glucokinase must interact with factors found in limiting concentration in the islet cell in order to become activated and engage in productive metabolic signaling.
Induction by Glucose of Genes Coding for Glycolytic Enzymes in a Pancreatic β-Cell Line (INS-1)
Journal of Biological Chemistry, 1997
Chronic elevation in glucose has pleiotropic effects on the pancreatic -cell including a high rate of insulin secretion at low glucose, -cell hypertrophy, and hyperplasia. These actions of glucose are expected to be associated with the modulation of the expression of a number of glucose-regulated genes that need to be identified. To further investigate the molecular mechanisms implicated in these adaptation processes to hyperglycemia, we have studied the regulation of genes encoding key glycolytic enzymes in the glucose-responsive -cell line INS-1. Glucose (from 5 to 25 mM) induced phosphofructokinase-1 (PFK-1) isoform C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4-fold), and L-pyruvate kinase (L-PK) (7-fold) mRNAs. In contrast the expression level of the glucokinase (Gk) and 6-phosphofructo-2-kinase transcripts remained unchanged. Following a 3-day exposure to elevated glucose, a similar induction was observed at the protein level for PFK-1 (isoforms C, M, and L), GAPDH, and L-PK, whereas M-PK expression only increased slightly. The study of the mechanism of GAPDH induction indicated that glucose increased the transcriptional rate of the GAPDH gene but that both transcriptional and post transcriptional effects contributed to GAPDH mRNA accumulation. 2-Deoxyglucose did not mimic the inductive effect of glucose, suggesting that increased glucose metabolism is involved in GAPDH gene induction. These changes in glycolytic enzyme expression were associated with a 2-3-fold increase in insulin secretion at low (2-5 mM) glucose. The metabolic activity of the cells was also elevated, as indicated by the reduction of the artificial electron acceptor 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium. A marked deposition of glycogen, which was readily mobilized upon lowering of the ambient glucose, and increased DNA replication were also observed in cells exposed to elevated glucose. The results suggest that a coordinated induction of key glycolytic enzymes as well as massive glycogen deposition are implicated in the adaptation process of the -cell to hyperglycemia to allow for chronically elevated glucose metabolism, which, in this particular fuel-sensitive cell, is linked to metabolic coupling factor production and cell activation.
Glucose Catabolism in Cancer Cells: Amplification of the Gene Encoding Type II Hexokinase1
Hexokinase type II is highly overexpressed in many cancer cells, where it plays a pivotal role in the high glycolytic phenotype. Here we demon strate by Southern blot analysis and fluorescence in situ hybridization (FISH) that in the rapidly growing rat AS-30D hepatoma cell line, en hanced hexokinase activity is associated with at least a 5-fold amplification of the type II gene relativeto normalhepatocytes. This amplificationis located chromosomally, extends to the whole gene, and most likely occurs at the site of the residentgene. No rearrangement of the gene could be detected. Therefore, overexpression of hexokinase type II in AS-30D hepatoma cells may be based, at least in part, on a stable gene amplifica tion. This is the first report describing the amplification of a hexokinase gene in a tumorcell line expressingthe high glycolyticphenotype.
Induction by Glucose of Genes Coding for Glycolytic Enzymes in a Pancreatic beta -Cell Line (INS-1)
Journal of Biological Chemistry, 1997
Chronic elevation in glucose has pleiotropic effects on the pancreatic -cell including a high rate of insulin secretion at low glucose, -cell hypertrophy, and hyperplasia. These actions of glucose are expected to be associated with the modulation of the expression of a number of glucose-regulated genes that need to be identified. To further investigate the molecular mechanisms implicated in these adaptation processes to hyperglycemia, we have studied the regulation of genes encoding key glycolytic enzymes in the glucose-responsive -cell line INS-1. Glucose (from 5 to 25 mM) induced phosphofructokinase-1 (PFK-1) isoform C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4-fold), and L-pyruvate kinase (L-PK) (7-fold) mRNAs. In contrast the expression level of the glucokinase (Gk) and 6-phosphofructo-2-kinase transcripts remained unchanged. Following a 3-day exposure to elevated glucose, a similar induction was observed at the protein level for PFK-1 (isoforms C, M, and L), GAPDH, and L-PK, whereas M-PK expression only increased slightly. The study of the mechanism of GAPDH induction indicated that glucose increased the transcriptional rate of the GAPDH gene but that both transcriptional and post transcriptional effects contributed to GAPDH mRNA accumulation. 2-Deoxyglucose did not mimic the inductive effect of glucose, suggesting that increased glucose metabolism is involved in GAPDH gene induction. These changes in glycolytic enzyme expression were associated with a 2-3-fold increase in insulin secretion at low (2-5 mM) glucose. The metabolic activity of the cells was also elevated, as indicated by the reduction of the artificial electron acceptor 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium. A marked deposition of glycogen, which was readily mobilized upon lowering of the ambient glucose, and increased DNA replication were also observed in cells exposed to elevated glucose. The results suggest that a coordinated induction of key glycolytic enzymes as well as massive glycogen deposition are implicated in the adaptation process of the -cell to hyperglycemia to allow for chronically elevated glucose metabolism, which, in this particular fuel-sensitive cell, is linked to metabolic coupling factor production and cell activation.