Negligible glucose-6-phosphatase activity in cultured astroglia

Jun Gotoh, Yoshiaki Itoh, Tang Yong Kuang, Michelle Cook, Mona J. Law, Louis Sokoloff

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

2-Deoxy[14C]glucose-6-phosphate (2-[14C]DG6-P) dephosphorylation and glucose-6-phosphatase (G-6Pase) activity were examined in cultured rat astrocytes under conditions similar to those generally used in assays of glucose utilization. Astrocytes were loaded with 2-[14C]DG6-P by preincubation for 15 min in medium containing 2 mM glucose and 50 μM 2- deoxy[14C]glucose (2-[14C]DG). The medium was then replaced with identical medium including 2 mM glucose but lacking 2-[14C]DG, and incubation was resumed for 5 min to diminish residual free 2-[14C]DG levels in the cells by either efflux or phosphorylation. The medium was again replaced with fresh 2-[14C]DG-free medium, and the incubation was continued for 5, 15, or 30 min. Intracellular and extracellular 14C contents were measured at each time point, and the distribution of 14C between 2- [14C]DG and 2-[14C]DG-6-P was characterized by paper chromatography. The results showed little if any hydrolysis of 2-[14C]DG-6-P or export of free 2-[14C]DG from cells to medium; there were slightly increasing losses of 2- [14C]DG and 2-[14C]DG-6-P into the medium with increasing incubation time, but they were in the same proportions found in the cells, suggesting they were derived from nonadherent or broken cells. Experiments carded out with medium lacking glucose during the assay for 2-deoxyglucose-6-phosphatase activity yielded similar results. Evidence for G-6-Pase activity was also sought by following the selective detritiation of glucose from the 2-C position when astrocytes were incubated with [2-3H]glucose and [U- 14C]glucose in the medium. No change in the 3H/14C ratio was found in incubations for as long as 15 min. These results indicate negligible G-6-Pase activity in cultured astrocytes.

Original languageEnglish (US)
Pages (from-to)1400-1408
Number of pages9
JournalJournal of Neurochemistry
Volume74
Issue number4
DOIs
StatePublished - 2000

Fingerprint

Glucose-6-Phosphatase
Astrocytes
Glucose
Assays
Paper Chromatography
Glucose-6-Phosphate
Phosphorylation
Deoxyglucose
Chromatography
Phosphoric Monoester Hydrolases
Rats
Hydrolysis

Keywords

  • 2-Deoxyglucose
  • Astrocytes
  • Glucose metabolism

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Gotoh, J., Itoh, Y., Kuang, T. Y., Cook, M., Law, M. J., & Sokoloff, L. (2000). Negligible glucose-6-phosphatase activity in cultured astroglia. Journal of Neurochemistry, 74(4), 1400-1408. https://doi.org/10.1046/j.1471-4159.2000.0741400.x

Negligible glucose-6-phosphatase activity in cultured astroglia. / Gotoh, Jun; Itoh, Yoshiaki; Kuang, Tang Yong; Cook, Michelle; Law, Mona J.; Sokoloff, Louis.

In: Journal of Neurochemistry, Vol. 74, No. 4, 2000, p. 1400-1408.

Research output: Contribution to journalArticle

Gotoh, J, Itoh, Y, Kuang, TY, Cook, M, Law, MJ & Sokoloff, L 2000, 'Negligible glucose-6-phosphatase activity in cultured astroglia', Journal of Neurochemistry, vol. 74, no. 4, pp. 1400-1408. https://doi.org/10.1046/j.1471-4159.2000.0741400.x
Gotoh, Jun ; Itoh, Yoshiaki ; Kuang, Tang Yong ; Cook, Michelle ; Law, Mona J. ; Sokoloff, Louis. / Negligible glucose-6-phosphatase activity in cultured astroglia. In: Journal of Neurochemistry. 2000 ; Vol. 74, No. 4. pp. 1400-1408.
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AU - Sokoloff, Louis

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N2 - 2-Deoxy[14C]glucose-6-phosphate (2-[14C]DG6-P) dephosphorylation and glucose-6-phosphatase (G-6Pase) activity were examined in cultured rat astrocytes under conditions similar to those generally used in assays of glucose utilization. Astrocytes were loaded with 2-[14C]DG6-P by preincubation for 15 min in medium containing 2 mM glucose and 50 μM 2- deoxy[14C]glucose (2-[14C]DG). The medium was then replaced with identical medium including 2 mM glucose but lacking 2-[14C]DG, and incubation was resumed for 5 min to diminish residual free 2-[14C]DG levels in the cells by either efflux or phosphorylation. The medium was again replaced with fresh 2-[14C]DG-free medium, and the incubation was continued for 5, 15, or 30 min. Intracellular and extracellular 14C contents were measured at each time point, and the distribution of 14C between 2- [14C]DG and 2-[14C]DG-6-P was characterized by paper chromatography. The results showed little if any hydrolysis of 2-[14C]DG-6-P or export of free 2-[14C]DG from cells to medium; there were slightly increasing losses of 2- [14C]DG and 2-[14C]DG-6-P into the medium with increasing incubation time, but they were in the same proportions found in the cells, suggesting they were derived from nonadherent or broken cells. Experiments carded out with medium lacking glucose during the assay for 2-deoxyglucose-6-phosphatase activity yielded similar results. Evidence for G-6-Pase activity was also sought by following the selective detritiation of glucose from the 2-C position when astrocytes were incubated with [2-3H]glucose and [U- 14C]glucose in the medium. No change in the 3H/14C ratio was found in incubations for as long as 15 min. These results indicate negligible G-6-Pase activity in cultured astrocytes.

AB - 2-Deoxy[14C]glucose-6-phosphate (2-[14C]DG6-P) dephosphorylation and glucose-6-phosphatase (G-6Pase) activity were examined in cultured rat astrocytes under conditions similar to those generally used in assays of glucose utilization. Astrocytes were loaded with 2-[14C]DG6-P by preincubation for 15 min in medium containing 2 mM glucose and 50 μM 2- deoxy[14C]glucose (2-[14C]DG). The medium was then replaced with identical medium including 2 mM glucose but lacking 2-[14C]DG, and incubation was resumed for 5 min to diminish residual free 2-[14C]DG levels in the cells by either efflux or phosphorylation. The medium was again replaced with fresh 2-[14C]DG-free medium, and the incubation was continued for 5, 15, or 30 min. Intracellular and extracellular 14C contents were measured at each time point, and the distribution of 14C between 2- [14C]DG and 2-[14C]DG-6-P was characterized by paper chromatography. The results showed little if any hydrolysis of 2-[14C]DG-6-P or export of free 2-[14C]DG from cells to medium; there were slightly increasing losses of 2- [14C]DG and 2-[14C]DG-6-P into the medium with increasing incubation time, but they were in the same proportions found in the cells, suggesting they were derived from nonadherent or broken cells. Experiments carded out with medium lacking glucose during the assay for 2-deoxyglucose-6-phosphatase activity yielded similar results. Evidence for G-6-Pase activity was also sought by following the selective detritiation of glucose from the 2-C position when astrocytes were incubated with [2-3H]glucose and [U- 14C]glucose in the medium. No change in the 3H/14C ratio was found in incubations for as long as 15 min. These results indicate negligible G-6-Pase activity in cultured astrocytes.

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