Measurement and modeling of glucose-6-phosphatase in pancreatic islets

I. R. Sweet, H. Najafi, G. Li, J. Grodberg and F. M. Matschinsky Department of Biochemistry, University of Pennsylvania, Philadelphia 19104, USA. In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the kinetic characte...

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Veröffentlicht in:American journal of physiology: endocrinology and metabolism 1997-04, Vol.272 (4), p.E696-E711
Hauptverfasser: Sweet, I. R, Najafi, H, Li, G, Grodberg, J, Matschinsky, F. M
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container_end_page E711
container_issue 4
container_start_page E696
container_title American journal of physiology: endocrinology and metabolism
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creator Sweet, I. R
Najafi, H
Li, G
Grodberg, J
Matschinsky, F. M
description I. R. Sweet, H. Najafi, G. Li, J. Grodberg and F. M. Matschinsky Department of Biochemistry, University of Pennsylvania, Philadelphia 19104, USA. In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the kinetic characteristics of glucokinase govern to a high degree the dose-response relationship between glucose and insulin release. Because glucose-6-phosphatase (G-6-Pase) opposes the action of glucokinase, it may have a regulatory role in the release of insulin in response to glucose if the enzyme is present in the beta-cells. A number of researchers have reported finding high levels of G-6-Pase in islets, but quantitation of its activity remains controversial, mainly because of difficulties in solubilizing a particulate enzyme. Therefore a method developed to measure functional glucose phosphorylation activity in intact brain was applied (Chi, M. M.-Y., M. E. Pusateri, J. G. Carter, B. J. Norris, D. B. McDougal, Jr., and O. H. Lowry. Anal. Biochem. 161: 508-513, 1987), and the rates of accumulation and disappearance of 2-deoxyglucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling. Islets were incubated in the presence of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incubation medium was replaced with medium containing no DG, but instead high levels of mannoheptulose as a blocker of phosphorylation. The content of DG-6-P in the islets was measured at strategic times during the protocol. As predicted by a mathematical model, DG-6-P accumulated in the presence of DG and decayed after its washout. Both of these results are consistent with islets containing dephosphorylation activity for this substrate. The kinetic curves were fit using a mathematical model, and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 micromol x g(-1) x min(-1). However, when the physiological effect of this amount of G-6-Pase activity was assessed by use of a model of glycolysis, it was found that the impact on glucose cycling and usage was insignificant. It was concluded that normal islets do contain measurable activity for dephosphorylating glucose 6-phosphate but that this enzymatic reaction does not play a role in glucose metabolism and sensing by the normal beta-cell.
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Therefore a method developed to measure functional glucose phosphorylation activity in intact brain was applied (Chi, M. M.-Y., M. E. Pusateri, J. G. Carter, B. J. Norris, D. B. McDougal, Jr., and O. H. Lowry. Anal. Biochem. 161: 508-513, 1987), and the rates of accumulation and disappearance of 2-deoxyglucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling. Islets were incubated in the presence of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incubation medium was replaced with medium containing no DG, but instead high levels of mannoheptulose as a blocker of phosphorylation. The content of DG-6-P in the islets was measured at strategic times during the protocol. As predicted by a mathematical model, DG-6-P accumulated in the presence of DG and decayed after its washout. Both of these results are consistent with islets containing dephosphorylation activity for this substrate. The kinetic curves were fit using a mathematical model, and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 micromol x g(-1) x min(-1). However, when the physiological effect of this amount of G-6-Pase activity was assessed by use of a model of glycolysis, it was found that the impact on glucose cycling and usage was insignificant. 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R</creatorcontrib><creatorcontrib>Najafi, H</creatorcontrib><creatorcontrib>Li, G</creatorcontrib><creatorcontrib>Grodberg, J</creatorcontrib><creatorcontrib>Matschinsky, F. M</creatorcontrib><title>Measurement and modeling of glucose-6-phosphatase in pancreatic islets</title><title>American journal of physiology: endocrinology and metabolism</title><addtitle>Am J Physiol</addtitle><description>I. R. Sweet, H. Najafi, G. Li, J. Grodberg and F. M. Matschinsky Department of Biochemistry, University of Pennsylvania, Philadelphia 19104, USA. In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the kinetic characteristics of glucokinase govern to a high degree the dose-response relationship between glucose and insulin release. Because glucose-6-phosphatase (G-6-Pase) opposes the action of glucokinase, it may have a regulatory role in the release of insulin in response to glucose if the enzyme is present in the beta-cells. A number of researchers have reported finding high levels of G-6-Pase in islets, but quantitation of its activity remains controversial, mainly because of difficulties in solubilizing a particulate enzyme. Therefore a method developed to measure functional glucose phosphorylation activity in intact brain was applied (Chi, M. M.-Y., M. E. Pusateri, J. G. Carter, B. J. Norris, D. B. McDougal, Jr., and O. H. Lowry. Anal. Biochem. 161: 508-513, 1987), and the rates of accumulation and disappearance of 2-deoxyglucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling. Islets were incubated in the presence of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incubation medium was replaced with medium containing no DG, but instead high levels of mannoheptulose as a blocker of phosphorylation. The content of DG-6-P in the islets was measured at strategic times during the protocol. As predicted by a mathematical model, DG-6-P accumulated in the presence of DG and decayed after its washout. Both of these results are consistent with islets containing dephosphorylation activity for this substrate. The kinetic curves were fit using a mathematical model, and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 micromol x g(-1) x min(-1). However, when the physiological effect of this amount of G-6-Pase activity was assessed by use of a model of glycolysis, it was found that the impact on glucose cycling and usage was insignificant. 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R</creatorcontrib><creatorcontrib>Najafi, H</creatorcontrib><creatorcontrib>Li, G</creatorcontrib><creatorcontrib>Grodberg, J</creatorcontrib><creatorcontrib>Matschinsky, F. M</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>American journal of physiology: endocrinology and metabolism</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sweet, I. R</au><au>Najafi, H</au><au>Li, G</au><au>Grodberg, J</au><au>Matschinsky, F. M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Measurement and modeling of glucose-6-phosphatase in pancreatic islets</atitle><jtitle>American journal of physiology: endocrinology and metabolism</jtitle><addtitle>Am J Physiol</addtitle><date>1997-04-01</date><risdate>1997</risdate><volume>272</volume><issue>4</issue><spage>E696</spage><epage>E711</epage><pages>E696-E711</pages><issn>0193-1849</issn><issn>0002-9513</issn><eissn>1522-1555</eissn><abstract>I. R. Sweet, H. Najafi, G. Li, J. Grodberg and F. M. Matschinsky Department of Biochemistry, University of Pennsylvania, Philadelphia 19104, USA. In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the kinetic characteristics of glucokinase govern to a high degree the dose-response relationship between glucose and insulin release. Because glucose-6-phosphatase (G-6-Pase) opposes the action of glucokinase, it may have a regulatory role in the release of insulin in response to glucose if the enzyme is present in the beta-cells. A number of researchers have reported finding high levels of G-6-Pase in islets, but quantitation of its activity remains controversial, mainly because of difficulties in solubilizing a particulate enzyme. Therefore a method developed to measure functional glucose phosphorylation activity in intact brain was applied (Chi, M. M.-Y., M. E. Pusateri, J. G. Carter, B. J. Norris, D. B. McDougal, Jr., and O. H. Lowry. Anal. Biochem. 161: 508-513, 1987), and the rates of accumulation and disappearance of 2-deoxyglucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling. Islets were incubated in the presence of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incubation medium was replaced with medium containing no DG, but instead high levels of mannoheptulose as a blocker of phosphorylation. The content of DG-6-P in the islets was measured at strategic times during the protocol. As predicted by a mathematical model, DG-6-P accumulated in the presence of DG and decayed after its washout. Both of these results are consistent with islets containing dephosphorylation activity for this substrate. The kinetic curves were fit using a mathematical model, and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 micromol x g(-1) x min(-1). However, when the physiological effect of this amount of G-6-Pase activity was assessed by use of a model of glycolysis, it was found that the impact on glucose cycling and usage was insignificant. It was concluded that normal islets do contain measurable activity for dephosphorylating glucose 6-phosphate but that this enzymatic reaction does not play a role in glucose metabolism and sensing by the normal beta-cell.</abstract><cop>United States</cop><pmid>9142893</pmid><doi>10.1152/ajpendo.1997.272.4.e696</doi></addata></record>
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source MEDLINE; American Physiological Society Paid; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection
subjects Animals
Computer Simulation
Deoxyglucose - pharmacology
Dose-Response Relationship, Drug
Glucose - pharmacology
Glucose-6-Phosphatase - metabolism
Glucose-6-Phosphate - analogs & derivatives
Glucose-6-Phosphate - metabolism
Homeostasis
In Vitro Techniques
Islets of Langerhans - enzymology
Male
Models, Biological
Perfusion
Phosphoric Monoester Hydrolases - metabolism
Rats
Rats, Wistar
Saccharomyces cerevisiae Proteins
title Measurement and modeling of glucose-6-phosphatase in pancreatic islets
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