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• W2044149898 abstract "Glucose is absolutely essential for the survival and function of the brain. In our current understanding, there is no endogenous glucose production in the brain, and it is totally dependent upon blood glucose. This glucose is generated between meals by the hydrolysis of glucose-6-phosphate (Glc-6-P) in the liver and the kidney. Recently, we reported a ubiquitously expressed Glc-6-P hydrolase, glucose-6-phosphatase-β (Glc-6-Pase-β), that can couple with the Glc-6-P transporter to hydrolyze Glc-6-P to glucose in the terminal stages of glycogenolysis and gluconeogenesis. Here we show that astrocytes, the main reservoir of brain glycogen, express both the Glc-6-Pase-β and Glc-6-P transporter activities and that these activities can couple to form an active Glc-6-Pase complex, suggesting that astrocytes may provide an endogenous source of brain glucose. Glucose is absolutely essential for the survival and function of the brain. In our current understanding, there is no endogenous glucose production in the brain, and it is totally dependent upon blood glucose. This glucose is generated between meals by the hydrolysis of glucose-6-phosphate (Glc-6-P) in the liver and the kidney. Recently, we reported a ubiquitously expressed Glc-6-P hydrolase, glucose-6-phosphatase-β (Glc-6-Pase-β), that can couple with the Glc-6-P transporter to hydrolyze Glc-6-P to glucose in the terminal stages of glycogenolysis and gluconeogenesis. Here we show that astrocytes, the main reservoir of brain glycogen, express both the Glc-6-Pase-β and Glc-6-P transporter activities and that these activities can couple to form an active Glc-6-Pase complex, suggesting that astrocytes may provide an endogenous source of brain glucose. Most tissues and organs in the body are not thought to be able to generate endogenous glucose. Therefore, between meals these tissues depend on glucose generated predominantly in the liver and kidney and distributed via the blood. The liver and the kidney are the primary organs responsible for interprandial blood glucose homeostasis. This homeostasis is dependent upon the activity of the glucose-6-phosphatase (Glc-6-Pase) 1The abbreviations used are: Glc-6-Pase, glucose-6-phosphatase; Glc-6-P, glucose-6-phosphate; Glc-PT, glucose-6-phosphate transporter; hGlc-6-Pase, human Glc-6-Pase; mGlc-6-Pase, mouse Glc-6-Pase; Ad, adenovirus; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; pfu, plaque-forming units. complex, which is comprised of a glucose-6-phosphate transporter (Glc-6-PT) and a Glc-6-Pase catalytic unit (reviewed in Refs. 1Chou J.Y. Matern D. Mansfield B.C. Chen Y.-T. Curr. Mol. Med. 2002; 2: 121-143Crossref PubMed Scopus (225) Google Scholar and 2Chen Y.-T. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1521-1551Google Scholar). The Glc-6-PT is a single copy gene (3Hiraiwa H. Pan C.-J. Lin B. Moses S.W. Chou J.Y. J. Biol. Chem. 1999; 274: 5532-5536Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 4Marcolongo P. Barone V. Priori G. Pirola B. Giglio S. Biasucci G. Zammarchi E. Parenti G. Burchell A. Benedetti A. Sorrentino V. FEBS Lett. 1998; 436: 247-250Crossref PubMed Scopus (49) Google Scholar, 5Gerin I. Veiga-da-Cunha M. Noel G. Van Schaftingen E. Gene. 1999; 227: 189-195Crossref PubMed Scopus (33) Google Scholar) that is expressed ubiquitously (6Lin B. Pan C.-J. Chou J.Y. Hum. Genet. 2000; 107: 526-529Crossref PubMed Scopus (26) Google Scholar). In contrast, until recently, Glc-6-Pase activity was considered restricted solely to the liver, kidney, and intestine (1Chou J.Y. Matern D. Mansfield B.C. Chen Y.-T. Curr. Mol. Med. 2002; 2: 121-143Crossref PubMed Scopus (225) Google Scholar, 2Chen Y.-T. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1521-1551Google Scholar). However, there is evidence that endogenous glucose production does occur outside of these tissues. In the genetic disease glycogen storage disease type Ia (1Chou J.Y. Matern D. Mansfield B.C. Chen Y.-T. Curr. Mol. Med. 2002; 2: 121-143Crossref PubMed Scopus (225) Google Scholar, 2Chen Y.-T. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1521-1551Google Scholar), where the liver/kidney/intestine Glc-6-Pase-α (G6PC) (7Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (319) Google Scholar, 8Shelly L.L. Lei K.-J. Pan C.-J. Sakata S.F. Ruppert S. Schutz G. Chou J.Y. J. Biol. Chem. 1993; 268: 21482-21485Abstract Full Text PDF PubMed Google Scholar) activity is deficient, patients are still capable of producing glucose (9Powell R.C. Wentworth S.M. Brandt I.K. Metabolism. 1981; 30: 443-450Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 10Tsalikian E. Simmons P. Gerich J.E. Howard C. Haymond M.W. Am. J. Physiol. 1984; 247: E513-E519PubMed Google Scholar, 11Collins J.E. Bartlett K. Leonard J.V. Aynsley-Green A. J. Inherit. Metab. Dis. 1990; 13: 195-206Crossref PubMed Scopus (13) Google Scholar), implying there are alternative pathways for endogenous glucose production. This led to the discovery of a second Glc-6-Pase activity, now called Glc-6-Pase-β (G6PC3 or UGRP) (12Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (72) Google Scholar, 13Guionie O. Clottes E. Stafford K. Burchell A. FEBS Lett. 2003; 551: 159-164Crossref PubMed Scopus (60) Google Scholar, 14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), which is widely expressed (12Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (72) Google Scholar). Glc-6-Pase-α (15Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), Glc-6-Pase-β (16Ghosh A. Shieh J.-J. Pan C.-J. Chou J.Y. J. Biol. Chem. 2004; 279: 12479-12483Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and Glc-6-PT (17Pan C.-J. Lin B. Chou J.Y. J. Biol. Chem. 1999; 274: 13865-13869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) are co-localized in the membrane of the endoplasmic reticulum (ER), embedded by multiple transmembrane domains. Glc-6-Pase-α is a 357-amino acid phosphohydrolase (7Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (319) Google Scholar, 8Shelly L.L. Lei K.-J. Pan C.-J. Sakata S.F. Ruppert S. Schutz G. Chou J.Y. J. Biol. Chem. 1993; 268: 21482-21485Abstract Full Text PDF PubMed Google Scholar) expressed primarily in the liver, kidney, and intestine (18Pan C.-J. Lei K.-J. Chen H. Ward J.M. Chou J.Y. Arch. Biochem. Biophys. 1998; 358: 17-24Crossref PubMed Scopus (59) Google Scholar, 19Nordlie R.C. Sukalski K.A. Martonosi A.N. The Enzymes of Biological Membranes. 2nd Ed. Plenum Press, New York1985: 349-398Crossref Google Scholar). Glc-6-Pase-β is a 346-amino acid phosphohydrolase (13Guionie O. Clottes E. Stafford K. Burchell A. FEBS Lett. 2003; 551: 159-164Crossref PubMed Scopus (60) Google Scholar, 14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) expressed ubiquitously (12Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (72) Google Scholar). Both exhibit similar active site structures, form similar covalently bound phosphoryl enzyme intermediates during catalysis (16Ghosh A. Shieh J.-J. Pan C.-J. Chou J.Y. J. Biol. Chem. 2004; 279: 12479-12483Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20Ghosh A. Shieh J.-J. Pan C.-J. Sun M.-S. Chou J.Y. J. Biol. Chem. 2002; 277: 32837-32842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and exhibit similar kinetic properties (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In the active Glc-6-Pase complex, Glc-6-PT transports cytoplasmic glucose-6-phosphate (Glc-6-P) across the ER membrane into the lumen where Glc-6-Pase, with its active site inside the lumen, hydrolyzes intra-lumenal Glc-6-P to glucose and phosphate (reviewed in Refs. 1Chou J.Y. Matern D. Mansfield B.C. Chen Y.-T. Curr. Mol. Med. 2002; 2: 121-143Crossref PubMed Scopus (225) Google Scholar and 2Chen Y.-T. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1521-1551Google Scholar). Alone, neither Glc-6-PT nor Glc-6-Pase-α have significant microsomal Glc-6-P transport activity, but when co-expressed they couple to significantly increase the activity of the Glc-6-Pase·Glc-6-PT complex (3Hiraiwa H. Pan C.-J. Lin B. Moses S.W. Chou J.Y. J. Biol. Chem. 1999; 274: 5532-5536Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). We have recently shown that Glc-6-Pase-β also has the ability to couple functionally with Glc-6-PT (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The discovery of Glc-6-Pase-β implies that non-hepatic tissues may be capable of endogenous glucose production through the activity of a Glc-6-Pase-β·Glc-6-PT complex. This is of particular interest, because the liver and kidney are not the sole sites of glycogen storage in the body. Indeed, the muscle is the major reservoir of body glycogen (21Baynes J. Dominiczak M.H. Baynes J. Medical Biochemistry. Mosby, London1999: 139-155Google Scholar, 22Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2004; 279: 26215-26219Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), and the brain, which expresses significant levels of both the Glc-6-PT (6Lin B. Pan C.-J. Chou J.Y. Hum. Genet. 2000; 107: 526-529Crossref PubMed Scopus (26) Google Scholar) and Glc-6-Pase-β (12Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (72) Google Scholar, 13Guionie O. Clottes E. Stafford K. Burchell A. FEBS Lett. 2003; 551: 159-164Crossref PubMed Scopus (60) Google Scholar), also stores glycogen. Moreover, glucose export from the brain has been demonstrated in children undergoing elective cardiopulmonary bypass surgery for congenital heart disease (23Eyre J.A. Stuart A.G. Forsyth R.J. Heaviside D. Bartlett K. Brain Res. 1994; 635: 349-352Crossref PubMed Scopus (36) Google Scholar), suggesting that the brain may be capable of endogenous glucose production. In the brain, the primary sites of glycogen storage are the astrocytes. Astrocytes are the most abundant glial cells in the central nervous system, responsible for regulating the external neuronal environment, responding to injury, and modulating neuronal growth and maturation (reviewed in Refs. 24Gruetter R. J. Neurosci. Res. 2003; 74: 179-183Crossref PubMed Scopus (156) Google Scholar and 25Brown A.M. J. Neurochem. 2004; 89: 537-552Crossref PubMed Scopus (249) Google Scholar). An acid-labile Glc-6-Pase-like activity (26Forsyth R.J. Bartlett K. Burchell A. Scott H.M. Eyre J.A. Biochem. J. 1993; 294: 145-151Crossref PubMed Scopus (43) Google Scholar) has been reported in astrocytes. However, the role of astrocyte glycogen in glucose production is controversial because the presence of a functional Glc-6-Pase complex has never been demonstrated in the brain. In this study, we show that brain astrocytes possess an active Glc-6-Pase-β·Glc-6-PT complex that can hydrolyze Glc-6-P to glucose, suggesting that astrocyte glycogen can be converted to glucose and may be a source of alternative energy in the neurons. Construction of Recombinant Adenoviral Mouse Glc-6-Pase-β— Mouse Glc-6-Pase-β (mGlc-6-Pase-β) containing a C-terminal FLAG peptide, DYKDDDDK (pSVL-mGlc-6-Pase-β-FLAG), was constructed by PCR from the pSVL-mGlc-6-Pase-β template (22Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2004; 279: 26215-26219Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) as described previously (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2004; 279: 26215-26219Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Recombinant adenoviruses containing mGlc-6-Pase-β (Ad-mGlc-6-Pase-β) or mGlc-6-Pase-β-FLAG (Ad-mGlc-6-Pase-β-FLAG) were generated by the Cre-lox recombination system (27Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar). The recombinant virus was plaque purified and amplified to produce viral stocks with titers of ∼5–10 × 109 plaque-forming units (pfu) per milliliter. Recombinant adenoviruses containing human Glc-6-Pase-β (Ad-hGlc-6-Pase-β), human Glc-6-Pase-α (Ad-hGlc-6-Pase-α), and human Glc-6-PT (Ad-hGlc-6-PT) have been described (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Expression in COS-1 Cells—COS-1 cells in 150-cm2 flasks were grown at 37 °C in HEPES-buffered Dulbecco's modified minimal essential medium supplemented with 4% fetal bovine serum. The cells were infected with Ad-mGlc-6-Pase-β or Ad-hGlc-6-Pase-α at various pfu/cell values and, after incubation at 37 °C for 48 h, either cell lysates or microsomes isolated from lysates were used for phosphohydrolase assays and Western blot analyses. For Glc-6-PT expression, COS-1 cells were infected with 50 pfu/cell Ad-hGlc-6-PT. For co-expression of Glc-6-PT and Glc-6-Pase, COS-1 cells were co-infected with 25 pfu/cell Ad-mGlc-6-Pase-β or Ad-hGlc-6-Pase-β and 50 pfu/cell Ad-hGlc-6-PT. For Glc-6-P uptake analysis, microsomes were isolated from lysates prepared after incubation at 37 °C for 24 h. Phosphohydrolase and Glc-6-P Uptake Analyses—Phosphohydrolase activity was determined essentially as described previously (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Glc-6-Pase-β in brain microsomes was assayed at the optimal temperature of 37 °C, and Glc-6-Pase-α in hepatic microsomes was assayed at 30 °C (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Reaction mixtures (100 μl) containing 50 mm cacodylate buffer, pH 6.5, 10 mm Glc-6-P, and appropriate amounts of microsomal preparations were incubated at either 37 or 30 °C for 10 min. Disrupted microsomal membranes were prepared by incubating intact microsomes in 0.2% deoxycholate for 20 min at 0 °C. Nonspecific phosphatase activity was estimated by pre-incubating disrupted microsomal preparations at pH 5, for 10 min at 37 °C to inactivate the acid-labile Glc-6-Pase-α and Glc-6-Pase-β. Glc-6-P uptake measurements were performed as described (3Hiraiwa H. Pan C.-J. Lin B. Moses S.W. Chou J.Y. J. Biol. Chem. 1999; 274: 5532-5536Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, microsomes (40 μg) were incubated in a reaction mixture (100 μl) containing 50 mm sodium cacodylate buffer, pH 6.5, 250 mm sucrose, and 0.2 mm [U-14C]Glc-6-P (50 μCi/μmol). The reaction was stopped at the appropriate time by filtering immediately through a nitrocellulose membrane (BA85; Schleicher & Schuell) and washing with an ice-cold solution containing 50 mm Tris-HCl, pH 7.4, and 250 mm sucrose. The radioactivity measured within the microsomes represents both the translocated substrate, [U-14C] Glc-6-P, and its hydrolytic product, [U-14C]glucose. Microsomes permeabilized with 0.2% deoxycholate to abolish Glc-6-P uptake were used as negative controls. Two to three independent experiments were conducted, and at least three Glc-6-P uptake studies were performed for each microsomal preparation. Statistical analysis using the unpaired t test was performed with the GraphPad Prism program (GraphPad Software, San Diego, CA). Data are presented as the mean ± S.E. Glc-6-Pase-α–/– and Glc-6-PT–/– Mice—Mice deficient in Glc-6-Pase-α (28Lei K.-J. Chen H. Pan C.-J. Ward J.M. Mosinger B. Lee E.J. Westphal H. Chou J.Y. Nat. Genet. 1996; 13: 203-209Crossref PubMed Scopus (189) Google Scholar) and Glc-6-PT (29Chen L.-Y. Shieh J.-J. Lin B. Pan C.-J. Gao J.-L. Murphy P.M. Roe T.F. Moses S. Ward J.M. Westphal H. Lee E.J. Mansfield B.C. Chou J.Y. Hum. Mol. Genet. 2003; 12: 2547-2558Crossref PubMed Scopus (75) Google Scholar) have been described. All animal studies were conducted under an animal protocol approved by the NICHD Animal Care and Use Committee. To maintain viability of the Glc-6-Pase-α–/– and Glc-6-PT–/– mice, glucose therapy consisting of intraperitoneal injection of 25–100 μl of 15% glucose every 12 h was initiated on the first post-natal day (29Chen L.-Y. Shieh J.-J. Lin B. Pan C.-J. Gao J.-L. Murphy P.M. Roe T.F. Moses S. Ward J.M. Westphal H. Lee E.J. Mansfield B.C. Chou J.Y. Hum. Mol. Genet. 2003; 12: 2547-2558Crossref PubMed Scopus (75) Google Scholar). Weaned mice were also given unrestricted access to mouse chow (Zeigler Bros., Inc., Gardners, PA). Microsomes were isolated from the brain and liver of 6 –7-week-old mice essentially as described (8Shelly L.L. Lei K.-J. Pan C.-J. Sakata S.F. Ruppert S. Schutz G. Chou J.Y. J. Biol. Chem. 1993; 268: 21482-21485Abstract Full Text PDF PubMed Google Scholar, 30Burchell A. Hume R. Burchell B. Clin. Chim. Acta. 1988; 173: 183-192Crossref PubMed Scopus (175) Google Scholar). Each microsomal preparation represents one individual mouse, and at least three independent microsomal preparations were used for each assay. Northern Blot and Western Blot Analyses—Total RNA was isolated by the guanidinium thiocyanate/cesium chloride method, fractionated by electrophoresis through 1.2% agarose gels containing 2.2 m formaldehyde, and transferred to a Nytran membrane by electroblotting. The filters were hybridized to a uniformly labeled mGlc-6-Pase-β, mGlc-6-PT, or β-actin riboprobe. For Western blot analysis, cell lysates or microsomal proteins were separated by electrophoresis through a 12% polyacrylamide-SDS gel and blotted onto polyvinylidene fluoride membranes (Millipore Co.). The membranes were incubated either with a monoclonal antibody against the FLAG epitope (Scientific Imaging Systems, Eastman Kodak), a polyclonal antibody against hGlc-6-Pase-β (22Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2004; 279: 26215-26219Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), or a rabbit anti-glial fibrillary acidic protein (GFAP) antibody (Affinity BioReagents, Inc., Golden, CO). The antigen-antibody complex was visualized as described previously (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2004; 279: 26215-26219Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Mouse Astrocytes in Primary Culture—Mouse astrocytes were prepared using the method of Pousset et al. (31Pousset F. Cremona S. Dantzer R. Kelley K. Parnet P. Glia. 1999; 26: 12-21Crossref PubMed Scopus (65) Google Scholar). Briefly, brains were dissected from 2–3-day-old wild-type, Glc-6-Pase-α–/–, or Glc-6-PT–/– pups, and the meningeal tissues were removed. The cortices were suspended in phosphate-buffered saline and flushed several times with a fire-polished Pasteur pipette. The resulting cell suspension was passed through a sterile nylon sieve (70-μm pore size; Falcon) to remove clumps and centrifuged at 1,200 rpm for 5 min to collect the cells. The cell pellet was resuspended in Dulbecco's modified minimal essential medium containing 20% heat-inactivated fetal bovine serum, seeded at a density of 9 × 104 cells/6-cm dishes, and then incubated at 37 °C in a humidified 5% CO2, 95% air atmosphere. Under these conditions, neurons do not survive the mechanical dissociation, and the low plating density prevents oligodendrocyte proliferation (31Pousset F. Cremona S. Dantzer R. Kelley K. Parnet P. Glia. 1999; 26: 12-21Crossref PubMed Scopus (65) Google Scholar). After 7 days in culture, the cells were incubated with fresh Dulbecco's modified minimal essential medium containing 10% fetal bovine serum, and this medium was changed weekly. After 21 days in culture, one set of cultures was fixed in 4% paraformaldehyde to stain for marker proteins. The second set of cultures was used to prepare cell lysates and microsomes for phosphohydrolase and Glc-6-P uptake assays and Western blot analyses. The purity of the astrocytes was determined by staining for GFAP (32Rutka J.T. Murakami M. Dirks P.B. Hubbard S.L. Becker L.E. Fukuyama K. Jung S. Tsugu A. Matsuzawa K. J. Neurosurg. 1997; 87: 420-430Crossref PubMed Scopus (146) Google Scholar). The cells were fixed for 10 min in 4% paraformaldehyde, incubated for 30 min at room temperature in TST buffer (0.05 m Tris-HCl, pH 7.5, 0.15 m NaCl, and 0.1% Triton-X-100) containing 1% bovine serum albumin (BSA) and 10% horse serum (TST-BSA), and then incubated overnight at 4 °C with a rabbit anti-GFAP antibody at 4 μg/ml in TST-BSA. Following three washes with TST buffer, the cells were then incubated with a biotinylated goat anti-rabbit IgG for 30 min, and the antigen-antibody complex was visualized with the Vectastatin Elite ABC kit (Vector Laboratories, Burlingame, CA). Replicates omitting the primary antibody or substituting the primary antibody with a preimmune rabbit serum were used as controls. A mouse monoclonal antibody against fibronectin (Sigma) and a rat monoclonal antibody against myelin basic protein (Sigma) were used to measure contamination of the cultures by fibroblasts and oligodendrocytes, respectively. Mouse Glc-6-Pase-β Is a Phosphohydrolase and Couples with Glc-6-PT to Form a Functional Glc-6-Pase Complex—The hGlc-6-Pase-β is a functional phosphohydrolase (13Guionie O. Clottes E. Stafford K. Burchell A. FEBS Lett. 2003; 551: 159-164Crossref PubMed Scopus (60) Google Scholar, 14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) that couples with the Glc-6-PT to form an active Glc-6-Pase complex (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, although hGlc-6-Pase-β (12Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (72) Google Scholar) and mGlc-6-Pase-β (33Boustead J.N. Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2004; 32: 33-53Crossref PubMed Scopus (19) Google Scholar) are very similar, both being 346-amino acid proteins with a conserved active site structure and an overall 84% amino acid sequence, mGlc-6-Pase-β is reported to lack activity (33Boustead J.N. Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2004; 32: 33-53Crossref PubMed Scopus (19) Google Scholar). To investigate whether mGlc-6-Pase-β might have a level of activity below the sensitivity of the reported assay, we examined the Glc-6-P hydrolytic activity of mGlc-6-Pase-β using a sensitive adenovirus-based expression system. Viral stocks of Ad-mGlc-6-Pase-β, Ad-mGlc-6-Pase-β-3FLAG, Ad-hGlc-6-Pase-β (or Ad-hGlc-6-Pase-β-3FLAG) (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and Ad-hGlc-6-Pase-α (or Ad-hGlc-6-Pase-α-3FLAG) (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) were used to infect monkey kidney COS-1 cells, and the resulting phosphohydrolase activities were assayed at pH 6.5 and 37 °C for Glc-6-Pase-β and pH 6.5 and 30 °C for Glc-6-Pase-α (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The Ad-hGlc-6-Pase-α-3FLAG-infected COS-1 lysates yield activities of 208.2 ± 7.9 and 347.6 ± 9.9 nmol/mg/min at a multiplicity of infection of 25 and 50 pfu/cell, respectively (Fig. 1A). The Ad-mGlc-6-Pase-β-3FLAG-infected COS-1 lysates have a lower but significant activity, showing a dose-dependent Glc-6-P hydrolytic activity that ranges from 11.8 ± 0.3 nmol/mg/min at a multiplicity of infection of 5 pfu/cell to 82.4 ± 5.1 nmol/mg/min at multiplicity of infection of 100 pfu/cell (Fig. 1A). In the same assay, the non-tagged protein Ad-mGlc-6-Pase-β has an activity identical to that of the tagged protein Ad-mGlc-6-Pase-β-3FLAG (data not shown). The mGlc-6-Pase-β activity is similar to the hGlc-6-Pase-β activity (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), both being 6-fold lower than that of hGlc-6-Pase-α (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) (Fig. 1A). Western analysis shows that the expression of mGlc-6-Pase-β and hGlc-6-Pase-α proteins correlates with enzymatic activity (Fig. 1A), as was shown previously for hGlc-6-Pase-β (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Previous studies have shown that both hGlc-6-Pase-α and hGlc-6-Pase-β are acid-labile and latent (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The mGlc-6-Pase-β is similarly acid-labile, losing >98% of Glc-6-P hydrolytic activity when Ad-mGlc-6-Pase-β-infected COS-1 microsomes are incubated at pH 5.0 for 10 min at 37 °C (Fig. 1B). The mGlc-6-Pase-β also exhibits a similar latency to that of hGlc-6-Pase-α and hGlc-6-Pase-β (Fig. 1B). Both human Glc-6-Pases also share a common pH optimum of 6.5, although the optimal temperatures for hGlc-6-Pase-α and hGlc-6-Pase-β differ, being 30 and 37 °C, respectively (14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Like hGlc-6-Pase-β, the optimal assay condition for mGlc-6-Pase-β is pH 6.5 and 37 °C (Fig. 1B). In the liver and kidney, Glc-6-P transport and hydrolysis are tightly coupled (28Lei K.-J. Chen H. Pan C.-J. Ward J.M. Mosinger B. Lee E.J. Westphal H. Chou J.Y. Nat. Genet. 1996; 13: 203-209Crossref PubMed Scopus (189) Google Scholar). The uptake and accumulation of Glc-6-P into the lumen of the ER is stimulated dramatically when hGlc-6-PT is co-expressed with either hGlc-6-Pase-α or hGlc-6-Pase-β (3Hiraiwa H. Pan C.-J. Lin B. Moses S.W. Chou J.Y. J. Biol. Chem. 1999; 274: 5532-5536Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The mGlc-6-Pase-β demonstrates a similar functional coupling to hGlc-6-PT (Fig. 1C). COS-1 cells infected with Ad-mGlc-6-Pase-β (or Ad-hGlc-6-Pase-β) have a very low level of microsomal Glc-6-P uptake activity (Fig. 1C), as was shown previously for hGlc-6-Pase-α (3Hiraiwa H. Pan C.-J. Lin B. Moses S.W. Chou J.Y. J. Biol. Chem. 1999; 274: 5532-5536Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Microsomal Glc-6-P transport activity was significantly increased in COS-1 cells infected with Ad-hGlc-6-PT alone (Fig. 1C), and the activity was markedly increased in cells co-infected with either Ad-hGlc-6-PT and Ad-mGlc-6-Pase-β or Ad-hGlc-6-PT and Ad-hGlc-6-Pase-β (Fig. 1C). The co-infected cultures also have identical time courses for microsomal Glc-6-P accumulation. Brain Expresses Active Glc-6-Pase-β—Glc-6-Pase-α is expressed primarily in the liver, kidney, and intestine (18Pan C.-J. Lei K.-J. Chen H. Ward J.M. Chou J.Y. Arch. Biochem. Biophys. 1998; 358: 17-24Crossref PubMed Scopus (59) Google Scholar, 19Nordlie R.C. Sukalski K.A. Martonosi A.N. The Enzymes of Biological Membranes. 2nd Ed. Plenum Press, New York1985: 349-398Crossref Google Scholar). Although both Glc-6-Pase-β (12Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (72) Google Scholar, 33Boustead J.N. Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2004; 32: 33-53Crossref PubMed Scopus (19) Google Scholar) and Glc-6-PT (6Lin B. Pan C.-J. Chou J.Y. Hum. Genet. 2000;" @default.
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• W2044149898 title "Brain Contains a Functional Glucose-6-Phosphatase Complex Capable of Endogenous Glucose Production" @default.
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