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 language | English (US) |
|---|---|
| Pages (from-to) | 1400-1408 |
| Number of pages | 9 |
| Journal | Journal of Neurochemistry |
| Volume | 74 |
| Issue number | 4 |
| DOIs | |
| State | Published - 2000 |
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Keywords
- 2-Deoxyglucose
- Astrocytes
- Glucose metabolism
ASJC Scopus subject areas
- Biochemistry
- Cellular and Molecular Neuroscience
Cite this
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 journal › Article
}
TY - JOUR
T1 - Negligible glucose-6-phosphatase activity in cultured astroglia
AU - Gotoh, Jun
AU - Itoh, Yoshiaki
AU - Kuang, Tang Yong
AU - Cook, Michelle
AU - Law, Mona J.
AU - Sokoloff, Louis
PY - 2000
Y1 - 2000
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.
KW - 2-Deoxyglucose
KW - Astrocytes
KW - Glucose metabolism
UR - http://www.scopus.com/inward/record.url?scp=0034023021&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034023021&partnerID=8YFLogxK
U2 - 10.1046/j.1471-4159.2000.0741400.x
DO - 10.1046/j.1471-4159.2000.0741400.x
M3 - Article
C2 - 10737595
AN - SCOPUS:0034023021
VL - 74
SP - 1400
EP - 1408
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
SN - 0022-3042
IS - 4
ER -