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 |
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Zusammenfassung: | 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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ISSN: | 0193-1849 0002-9513 1522-1555 |
DOI: | 10.1152/ajpendo.1997.272.4.e696 |