Liver glucose-6-phosphatase activity is not modulated by physiological intracellular Ca2+ concentrations

Fulceri, Rosella; Bellomo, Giorgio; Gamberucci, Alessandra; Benedetti, Angelo

doi:10.1042/bj2750805

Cited by 8 publications

(5 citation statements)

References 19 publications

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“…To this end, the time-dependent loss of the microsome-associated radioactivity was evaluated in vanadatetreated microsomes pre-equilibrated with G-6-P (0.1 and 1.0 mm plus [14C]G-6-P as a tracer) and diluted in a G-6-P-free medium. It is unlikely that 14C associated with microsomes might be accounted for by [14C]glucose already present in the [14C]G-6-P solution, or derived from some hydrolysis of [14C]G-6-P, because [14C]glucose in the incubation medium never exceeded 3 % of total radioactivity (as measured at the beginning and at the end of the experiments after the removal of [14C]G-6-P by barium precipitation [31]; result not shown).…”

Section: Resultsmentioning

confidence: 99%

Permeability of rat liver microsomal membrane to glucose 6-phosphate

Fulceri

Bellomo

Gamberucci

et al. 1992

Biochemical Journal

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Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer revealed the following. (1) The increase in extravesicular osmolality by addition of glucose 6-phosphate or mannose 6-phosphate (25 mM each) caused a rapid shrinking of microsomal vesicles. After shrinkage, a rapid swelling phase (t1/2 approx. 22 s) was present with glucose 6-phosphate but absent with mannose 6-phosphate, indicating that the former had entered microsomal vesicles, but the latter had not. (2) Almost identical results were obtained in the absence of any glucose 6-phosphate hydrolysis, i.e. with microsomes pre-treated with 100 microM-vanadate. (3) The anion-channel blocker 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) suppressed the glucose 6-phosphate-induced swelling phase. (4) The swelling phase was more prolonged as the glucose 6-phosphate concentration increased (t1/2 = 16 +/- 3, 22 +/- 3 and 35 +/- 4 s with 25 mM, 37.5 mM- and 50 mM-glucose 6-phosphate respectively). The behaviour of glucose-6-phosphatase activity of intact and disrupted microsomes measured in the presence of high concentrations (less than 30 mM) of substrate also indicated the saturation of the glucose 6-phosphate permeation system by extravesicular concentrations of glucose 6-phosphate higher than 20-30 mM. Additional experiments showed that vanadate-treated microsomes pre-equilibrated with 0.1 mM- and 1.0 mM-glucose 6-phosphate (and [1-14C]glucose 6-phosphate as a tracer) rapidly (t1/2 less than 20 s) released [1-14C]glucose 6-phosphate when diluted in a glucose 6-phosphate-free medium. The efflux of [1-14C]glucose 6-phosphate was largely prevented by DIDS, allowing an evaluation of the intravesicular space of glucose 6-phosphate of approx. 1.0 microliter/mg of microsomal protein.

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Section: Resultsmentioning

confidence: 99%

Permeability of rat liver microsomal membrane to glucose 6-phosphate

Fulceri

Bellomo

Gamberucci

et al. 1992

Biochemical Journal

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“…After mixing, 0.5 ml of a saturated solution of Ba(OH) 2 were added, and after vortexing the tubes were centrifuged for 2 min at 14,000 rpm in an Eppendorf centrifuge. Aliquots of the supernatant were counted for each sample, and relative amounts of [ 3 H]DG formed were determined (19). (20).…”

Section: Methodsmentioning

confidence: 99%

Cellular Release of [18F]2-Fluoro-2-deoxyglucose as a Function of the Glucose-6-phosphatase Enzyme System

Caracò¹,

Aloj²,

Chen³

et al. 2000

Journal of Biological Chemistry

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[ 18 F]-2-Fluoro-2-deoxyglucose (FDG) is a glucose analog currently utilized for positron emission tomography imaging studies in humans. FDG taken up by the liver is rapidly released. This property is attributed to elevated glucose-6-phosphatase (Glc-6-Pase) activity. To characterize this issue we studied the relationship between Glc-6-Pase activity and FDG release kinetics in a cell culture system. We overexpressed the Glc-6-Pase catalytic unit in a Glc-6-Pase-deficient mouse hepatocyte (Ho-15) and in A431 tumor cell lines. Glc-6-Pase enzyme activity and FDG release rates were determined in cells transfected with the Glc-6-Pase gene (Ho-15-D3 and A431-AC3), in mock-transfected cells of both cell lines, and in wild-type mouse hepatocytes (WT10) as control. Although the highest level of Glc-6-Pase activity was measured in A431-AC3, Ho-15-D3 cells showed much faster FDG release rates. The faster FDG release correlated with the level of glucose 6-phosphate transporter (Glc-6-PT) mRNA, which was found to be expressed at higher levels in Ho-15 compared with A431 cells. Overexpression of Glc-6-PT in A431-AC3 produced a dramatic increase in FDG release compared with control cells. This study gives the first direct evidence that activity of the Glc-6-Pase complex can be quantified in vivo by measuring FDG release. Adequate levels of Glc-6-Pase catalytic unit and Glc-6-PT are required for this function. FDG-positron emission tomography may be utilized to evaluate functional status of the Glc-6-Pase complex.

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“…Parallel filters were treated with ZnSO 4 -Ba(OH) 2 to separate [ 14 C]glucose from [ 14 C]glucose-6-phosphate, and labeled glucose-6-phosphate and glucose were recovered, respectively, in the pellet and supernatant, after centrifugation (43). Briefly, washed filters were transferred into tubes containing 0.3 ml of 0.2 M ZnSO 4 and were pushed to the bottom.…”

Section: Methodsmentioning

confidence: 99%

“…Assay of Glucose-6-phosphatase-Glucose-6-phosphatase activity was measured after 5 min of incubation in buffer A at 22°C on the basis of D-[ 14 C(U)]glucose production from D-[ 14 C(U)]glucose-6-phosphate according to (43). At high substrate concentrations (30 mM), the enzyme activity was also evaluated by measuring glucose production with a glucose (Trinder) kit (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Demonstration of a Metabolically Active Glucose-6-phosphate Pool in the Lumen of Liver Microsomal Vesicles

Bánhegyi

Marcolongo

Fulceri

et al. 1997

Journal of Biological Chemistry

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Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.

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Liver glucose-6-phosphatase activity is not modulated by physiological intracellular Ca2+ concentrations

Cited by 8 publications

References 19 publications

Permeability of rat liver microsomal membrane to glucose 6-phosphate

Permeability of rat liver microsomal membrane to glucose 6-phosphate

Cellular Release of [18F]2-Fluoro-2-deoxyglucose as a Function of the Glucose-6-phosphatase Enzyme System

Demonstration of a Metabolically Active Glucose-6-phosphate Pool in the Lumen of Liver Microsomal Vesicles

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