Negligible Glucose‐6‐Phosphatase Activity in Cultured Astroglia

2‐Deoxy[ 14 C]glucose‐6‐phosphate(2‐[ 14 C]DG‐6‐P) dephosphorylation and glucose‐6‐phosphatase(G‐6‐Pase) activity were examined in cultured rat astrocytes under conditionssimilar to those generally used in assays of glucose utilization. Astrocyteswere loaded with 2‐[ 14 C]DG‐6‐P by preincubation for 15 min inmedium containing 2 m M glucose and 50 μ M 2‐deoxy[ 14 C]glucose (2‐[ 14 C]DG). The medium was thenreplaced with identical medium including 2 m M glucose but lacking2‐[ 14 C]DG, and incubation was resumed for 5 min to diminishresidual free 2‐[ 14 C]DG levels in the cells by either efflux orphosphorylation. The medium was again replaced with fresh2‐[ 14 C]DG‐free medium, and the incubation was continued for 5, 15,or 30 min. Intracellular and extracellular 14 C contents weremeasured at each time point, and the distribution of 14 C between2‐[ 14 C]DG and 2‐[ 14 C]DG‐6‐P was characterized by paperchromatography. The results showed little if any hydrolysis of2‐[ 14 C]DG‐6‐P or export of free 2‐[ 14 C]DG from cells tomedium; there were slightly increasing losses of 2‐[ 14 C]DG and2‐[ 14 C]DG‐6‐P into the medium with increasing incubation time, butthey were in the same proportions found in the cells, suggesting they werederived from nonadherent or broken cells. Experiments carried out with mediumlacking glucose during the assay for 2‐deoxyglucose‐6‐phosphatase activityyielded similar results. Evidence for G‐6‐Pase activity was also sought byfollowing the selective detritiation of glucose from the 2‐C position whenastrocytes were incubated with [2‐ 3 H]glucose and[U‐ 14 C]glucose in the medium. No change in the 3 H/ 14 C ratio was found in incubations for as long as 15 min. These results indicate negligible G‐6‐Pase activity in cultured astrocytes.