JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M410894200 on January 20, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11114-11119, March 25, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11114    most recent
M410894200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ghosh, A.
Right arrow Articles by Chou, J. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ghosh, A.
Right arrow Articles by Chou, J. Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Brain Contains a Functional Glucose-6-Phosphatase Complex Capable of Endogenous Glucose Production*

Abhijit Ghosh, Yuk Yin Cheung, Brian C. Mansfield, and Janice Yang Chou{ddagger}

From the Section on Cellular Differentiation, Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, September 22, 2004 , and in revised form, January 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{beta} (Glc-6-Pase-{beta}), 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-{beta} 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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)1 complex, which is comprised of a glucose-6-phosphate transporter (Glc-6-PT) and a Glc-6-Pase catalytic unit (reviewed in Refs. 1 and 2). The Glc-6-PT is a single copy gene (3–5) that is expressed ubiquitously (6). In contrast, until recently, Glc-6-Pase activity was considered restricted solely to the liver, kidney, and intestine (1, 2). However, there is evidence that endogenous glucose production does occur outside of these tissues. In the genetic disease glycogen storage disease type Ia (1, 2), where the liver/kidney/intestine Glc-6-Pase-{alpha} (G6PC) (7, 8) activity is deficient, patients are still capable of producing glucose (9–11), 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-{beta} (G6PC3 or UGRP) (12–14), which is widely expressed (12).

Glc-6-Pase-{alpha} (15), Glc-6-Pase-{beta} (16), and Glc-6-PT (17) are co-localized in the membrane of the endoplasmic reticulum (ER), embedded by multiple transmembrane domains. Glc-6-Pase-{alpha} is a 357-amino acid phosphohydrolase (7, 8) expressed primarily in the liver, kidney, and intestine (18, 19). Glc-6-Pase-{beta} is a 346-amino acid phosphohydrolase (13, 14) expressed ubiquitously (12). Both exhibit similar active site structures, form similar covalently bound phosphoryl enzyme intermediates during catalysis (16, 20), and exhibit similar kinetic properties (14). 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. 1 and 2). Alone, neither Glc-6-PT nor Glc-6-Pase-{alpha} 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 (3). We have recently shown that Glc-6-Pase-{beta} also has the ability to couple functionally with Glc-6-PT (14).

The discovery of Glc-6-Pase-{beta} implies that non-hepatic tissues may be capable of endogenous glucose production through the activity of a Glc-6-Pase-{beta}·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 (21, 22), and the brain, which expresses significant levels of both the Glc-6-PT (6) and Glc-6-Pase-{beta} (12, 13), also stores glycogen. Moreover, glucose export from the brain has been demonstrated in children undergoing elective cardiopulmonary bypass surgery for congenital heart disease (23), 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. 24 and 25). An acid-labile Glc-6-Pase-like activity (26) 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-{beta}·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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Recombinant Adenoviral Mouse Glc-6-Pase-{beta}— Mouse Glc-6-Pase-{beta} (mGlc-6-Pase-{beta}) containing a C-terminal FLAG peptide, DYKDDDDK (pSVL-mGlc-6-Pase-{beta}-FLAG), was constructed by PCR from the pSVL-mGlc-6-Pase-{beta} template (22) as described previously (14, 22). Recombinant adenoviruses containing mGlc-6-Pase-{beta} (Ad-mGlc-6-Pase-{beta}) or mGlc-6-Pase-{beta}-FLAG (Ad-mGlc-6-Pase-{beta}-FLAG) were generated by the Cre-lox recombination system (27). The recombinant virus was plaque purified and amplified to produce viral stocks with titers of ~5–10 x 109 plaque-forming units (pfu) per milliliter. Recombinant adenoviruses containing human Glc-6-Pase-{beta} (Ad-hGlc-6-Pase-{beta}), human Glc-6-Pase-{alpha} (Ad-hGlc-6-Pase-{alpha}), and human Glc-6-PT (Ad-hGlc-6-PT) have been described (14).

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-{beta} or Ad-hGlc-6-Pase-{alpha} 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-{beta} or Ad-hGlc-6-Pase-{beta} 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 (14). Glc-6-Pase-{beta} in brain microsomes was assayed at the optimal temperature of 37 °C, and Glc-6-Pase-{alpha} in hepatic microsomes was assayed at 30 °C (14). 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-{alpha} and Glc-6-Pase-{beta}.

Glc-6-P uptake measurements were performed as described (3, 14). 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-{alpha}–/– and Glc-6-PT–/– Mice—Mice deficient in Glc-6-Pase-{alpha} (28) and Glc-6-PT (29) 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-{alpha}–/– 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 (29). 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 (8, 30). 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-{beta}, mGlc-6-PT, or {beta}-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-{beta} (22), or a rabbit anti-glial fibrillary acidic protein (GFAP) antibody (Affinity BioReagents, Inc., Golden, CO). The antigen-antibody complex was visualized as described previously (14, 22).

Mouse Astrocytes in Primary Culture—Mouse astrocytes were prepared using the method of Pousset et al. (31). Briefly, brains were dissected from 2–3-day-old wild-type, Glc-6-Pase-{alpha}–/–, 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 x 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 (31). 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 (32). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mouse Glc-6-Pase-{beta} Is a Phosphohydrolase and Couples with Glc-6-PT to Form a Functional Glc-6-Pase Complex—The hGlc-6-Pase-{beta} is a functional phosphohydrolase (13, 14) that couples with the Glc-6-PT to form an active Glc-6-Pase complex (14). However, although hGlc-6-Pase-{beta} (12) and mGlc-6-Pase-{beta} (33) 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-{beta} is reported to lack activity (33). To investigate whether mGlc-6-Pase-{beta} 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-{beta} using a sensitive adenovirus-based expression system. Viral stocks of Ad-mGlc-6-Pase-{beta}, Ad-mGlc-6-Pase-{beta}-3FLAG, Ad-hGlc-6-Pase-{beta} (or Ad-hGlc-6-Pase-{beta}-3FLAG) (14), and Ad-hGlc-6-Pase-{alpha} (or Ad-hGlc-6-Pase-{alpha}-3FLAG) (14) 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-{beta} and pH 6.5 and 30 °C for Glc-6-Pase-{alpha} (14). The Ad-hGlc-6-Pase-{alpha}-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-{beta}-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-{beta} has an activity identical to that of the tagged protein Ad-mGlc-6-Pase-{beta}-3FLAG (data not shown). The mGlc-6-Pase-{beta} activity is similar to the hGlc-6-Pase-{beta} activity (14), both being 6-fold lower than that of hGlc-6-Pase-{alpha} (14) (Fig. 1A). Western analysis shows that the expression of mGlc-6-Pase-{beta} and hGlc-6-Pase-{alpha} proteins correlates with enzymatic activity (Fig. 1A), as was shown previously for hGlc-6-Pase-{beta} (14).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 1.
mGlc-6-Pase-{beta} is a Glc-6-P phosphohydrolase. A, expression and activity of Ad-mGlc-6-Pase-{beta}-3FLAG and Ad-hGlc-6-Pase-{alpha}-3FLAG in infected COS-1 lysates. COS-1 cells were infected with Ad-mGlc-6-Pase-{beta}-3FLAG or Ad-hGlc-6-Pase-{alpha}-3FLAG at various multiplicity of infection (MOI) values. Expression of Ad-mGlc-6-Pase-{beta}-3FLAG and Ad-hGlc-6-Pase-{alpha}-3FLAG was detected by Western blot analysis using an anti-FLAG monoclonal antibody. The Glc-6-P phosphohydrolase activity was assayed at pH 6.5 and 37 °C for mGlc-6-Pase-{beta} and pH 6.5 and 30 °C for hGlc-6-Pase-{alpha}. The background activity of mock-infected cultures (5.4 ± 0.2 nmol/mg/min for mGlc-6-Pase-{beta} and 8.2 ± 0.5 nmol/mg/min for hGlc-6-Pase-{alpha}) was subtracted from the data presented. The Ad-mGlc-6-Pase-{beta} has an equivalent activity. B, comparison of the Glc-6-P phosphohydrolase activity of mGlc-6-Pase-{beta}, hGlc-6-Pase-{beta}, and hGlc-6-Pase-{alpha}. Microsomes isolated from COS-1 cells infected with the appropriate recombinant adenoviral construct at 50 pfu/cell were assayed at pH 6.5 and 37 °C for Glc-6-Pase-{beta} and pH 6.5 and 30 °C for Glc-6-Pase-{alpha}. Acid stability was determined by assaying Glc-6-P phosphohydrolase activity in deoxycholate-disrupted (0.2%) microsomes before and after preincubation at pH 5.0 for 10 min at 37 °C. Latency was measures as the rate of hydrolysis of mannose-6-phosphate in intact (I) versus detergent-disrupted (D) microsomes and defined as (1 – I/D) x 100 (19). The pH and temperature optima were determined by assaying Glc-6-P phosphohydrolase activity in deoxycholate-disrupted microsomes. C, mGlc-6-Pase-{beta} couples with Glc-6-PT to mediate microsomal Glc-6-P uptake. Microsomal Glc-6-P uptake activity in COS-1 cells infected with Ad-mGlc-6-Pase-{beta} (25 pfu/cell) ({triangledown}) or Ad-hGlc-6-PT (50 pfu/cell) ({blacktriangledown}), co-infected with Ad-mGlc-6-Pase-{beta} (25 pfu/cell) and Ad-hGlc-6-PT (50 pfu/cell) ({circ}), or co-infected with Ad-hGlc-6-Pase-{beta} (25 pfu/cell) and Ad-hGlc-6-PT (50 pfu/cell) (•) was measured over time. The radioactivity accumulated in the lumen of the ER consists of both [U-14C]Glc-6-P and [U-14C]glucose.

 
Previous studies have shown that both hGlc-6-Pase-{alpha} and hGlc-6-Pase-{beta} are acid-labile and latent (14). The mGlc-6-Pase-{beta} is similarly acid-labile, losing >98% of Glc-6-P hydrolytic activity when Ad-mGlc-6-Pase-{beta}-infected COS-1 microsomes are incubated at pH 5.0 for 10 min at 37 °C (Fig. 1B). The mGlc-6-Pase-{beta} also exhibits a similar latency to that of hGlc-6-Pase-{alpha} and hGlc-6-Pase-{beta} (Fig. 1B). Both human Glc-6-Pases also share a common pH optimum of 6.5, although the optimal temperatures for hGlc-6-Pase-{alpha} and hGlc-6-Pase-{beta} differ, being 30 and 37 °C, respectively (14). Like hGlc-6-Pase-{beta}, the optimal assay condition for mGlc-6-Pase-{beta} is pH 6.5 and 37 °C (Fig. 1B).

In the liver and kidney, Glc-6-P transport and hydrolysis are tightly coupled (28). 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-{alpha} or hGlc-6-Pase-{beta} (3, 14). The mGlc-6-Pase-{beta} demonstrates a similar functional coupling to hGlc-6-PT (Fig. 1C). COS-1 cells infected with Ad-mGlc-6-Pase-{beta} (or Ad-hGlc-6-Pase-{beta}) have a very low level of microsomal Glc-6-P uptake activity (Fig. 1C), as was shown previously for hGlc-6-Pase-{alpha} (3). 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-{beta} or Ad-hGlc-6-PT and Ad-hGlc-6-Pase-{beta} (Fig. 1C). The co-infected cultures also have identical time courses for microsomal Glc-6-P accumulation.

Brain Expresses Active Glc-6-Pase-{beta}—Glc-6-Pase-{alpha} is expressed primarily in the liver, kidney, and intestine (18, 19). Although both Glc-6-Pase-{beta} (12, 33) and Glc-6-PT (6) are expressed ubiquitously, only the brain, heart, skeletal muscle, and kidney express significant levels of both proteins (6, 12, 33). In the wild-type mice, expression of the Glc-6-Pase-{beta} transcript is slightly higher in the brain than in the kidney, whereas the expression of the Glc-6-PT transcript is substantially more elevated in the kidney than in the brain (Fig. 2A). Western blot analysis confirms the presence of the Glc-6-Pase-{beta} protein in the brains of wild-type mice (Fig. 2B). Western blot analysis of Glc-6-Pase-{alpha}–/– mouse brain, which lacks the Glc-6-Pase-{alpha} gene (28), shows a similar level of Glc-6-Pase-{beta} expression, confirming the specificity of the Glc-6-Pase-{beta} detection (Fig. 2B). As expected, the Glc-6-PT–/– mouse brain (29) contains a similar level of the Glc-6-Pase-{beta} protein as that of the wild-type mouse brain (Fig. 2B).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 2.
Expression of Glc-6-Pase-{beta} and Glc-6-PT in the brain. A, Northern blot analysis. Total RNA was isolated from the brain and kidney of wild-type mice and separated on a formaldehyde-agarose gel, and duplicate blots were hybridized with a riboprobe for Glc-6-Pase-{beta}, Glc-6-PT, or {beta}-actin. B, Western blot analysis. Glc-6-Pase-{beta} in microsomes isolated from the brains of Glc-6-Pase-{alpha}–/–, Glc-6-PT–/–, and wild-type mice were detected using an anti-hGlc-6-Pase-{beta} antibody (22) as described under "Experimental Procedures." The lane marked mGlc-6-Pase-{beta} represents protein from Ad-mGlc-6-Pase-{beta}-infected COS-1 lysates.

 
In contrast to nonspecific phosphohydrolases, Glc-6-Pase has a characteristic acid-labile profile (34). Therefore, to measure the specific Glc-6-P hydrolase activity in the brain, we performed two measurements, one on disrupted brain microsomes to measure total phosphatase activity and the other on disrupted microsomes pre-incubated at pH 5.0 and 37 °C for 10 min to measure the residual acid-resistant nonspecific phosphatases. The difference between these measurements reflects the acid-sensitive Glc-6-Pase-specific activity. The acid-sensitive Glc-6-P hydrolase activity in microsomes isolated from the brain of wild-type mice is 10.07 ± 0.84 nmol/mg/min (Table I), which represents 60% of the total phosphohydrolase activity. The brain Glc-6-P hydrolase activity is ~5% of the activity of the liver at 217.2 ± 6.0 nmol/mg/min (22). A previous report (26) ascribed 40% of the activity to acid-sensitive Glc-6-Pase when assayed at pH 6.5 and 30 °C. Our findings are reasonably consistent with this report, given that the initial finding was assayed at a temperature suboptimal for Glc-6-Pase-{beta} (Fig. 1B).


View this table:
[in this window]
[in a new window]
  		TABLE I
Brain acid-sensitive Glc-6-P phosphohydrolase activity and inhibition by vanadate

Glc-6-P phosphohydrolase activity was assayed using three independent brain microsomal preparations and performed with two measurements, namely one on disrupted microsomes to measure total phosphatase activity and the other on disrupted microsomes pre-incubated at pH 5.0 and 37 °C for 10 min to measure the residual acid-resistant non-specific phosphatases. The difference between these measurements reflects the acid-sensitive Glc-6-Pase specific activity. Data are present as the mean ± S.E. The concentration of vanadate for 50% inhibition of Glc-6-P phosphohydrolase activity ([vanadate]0.5) was determined in deoxycholate-disrupted microsomes as described previously (14).

 
To exclude the possibility that a low level of expression of Glc-6-Pase-{alpha} occurs in the brain and is more readily measured at the higher temperature we used to assay Glc-6-Pase-{beta}, we examined the Glc-6-P hydrolytic activity of brain microsomes from Glc-6-Pase-{alpha}–/– mice. The Glc-6-Pase-{alpha}–/– brain has an activity identical to that of the wild-type brain, implying that Glc-6-Pase-{alpha} is not expressed in the brain (Table I). As expected, Glc-6-PT–/– brain microsomes have an identical activity as that of the wild-type brain (Table I). To further support the identification of the acid-labile brain activity as Glc-6-Pase-{beta}, the vanadate sensitivity (Table I) of the Glc-6-Pase in the brain was examined. The hepatic hGlc-6-Pase-{alpha} activity is inhibited 50% at a vanadate concentration of 3.1 mM ([vanadate]0.5 = 3.1 mM) (14), whereas the hGlc-6-Pase-{beta} has a sensitivity of [vanadate]0.5 equal to 1.4 mM (14). Consistent with this finding, the mouse brain Glc-6-Pase activity is similar to the hGlc-6-Pase-{beta} activity, with a [vanadate]0.5 of 1.3–1.34 mM (Table I).

Brain Possesses an Active Glc-6-Pase Complex—In vivo, Glc-6-PT must couple functionally with Glc-6-Pase to form an efficient Glc-6-Pase complex (28). A comparison of Glc-6-P uptake in wild-type, Glc-6-Pase-{alpha}–/–, and Glc-6-PT–/– mouse brains shows that whereas the time courses of Glc-6-P uptake activity in the intact microsomes of wild-type and Glc-6-Pase-{alpha}–/– mice are identical, the Glc-6-PT–/– mice do show a markedly reduced Glc-6-P accumulation (Fig. 3A), consistent with the absence of Glc-6-PT in these mice (29). The brain Glc-6-P uptake activity is not attenuated in Glc-6-Pase-{alpha}–/– mice, consistent with the brain Glc-6-Pase-{beta} coupling with Glc-6-PT. Knock-out of Glc-6-PT in Glc-6-PT–/– mice results in ~82% loss of brain activity (Fig. 3B), confirming the importance of Glc-6-PT in both tissues. Because ~18% of wild-type Glc-6-P uptake activity does remain in the brain of Glc-6-PT-deficient mice, the possibility that there is another minor Glc-6-P transport protein in the brain cannot be excluded.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 3.
The Glc-6-Pase-{beta} complex in the brain is functional. Glc-6-P uptake assays were performed using microsomes isolated from the brain or liver of wild-type, Glc-6-Pase-{alpha}–/–, and Glc-6-PT–/– mice as described under "Experimental Procedures." A, time course of Glc-6-P uptake activity in intact microsomes isolated from the brain of wild-type ({circ}), Glc-6-Pase-{alpha}–/– (•), or Glc-6-PT–/– ({blacktriangledown}) mice. B, microsomal Glc-6-P uptake activity. The radioactivity accumulated in the lumen of the ER consists of both [U-14C]Glc-6-P and [U-14C]glucose. The results are given as mean ± S.E.

 
Brain Astrocytes Possess an Active Glc-6-Pase-{beta}·Glc-6-PT Complex—In the brain, astrocytes constitute >50% of the cell mass and are the only cells that contain substantial amounts of glycogen (reviewed in Ref. 35). If astrocytes are a site of expression of Glc-6-Pase-{beta} and Glc-6-PT, they may be capable of providing an endogenous source of glucose for the brain. To examine this possibility we isolated primary mouse astrocytes from 2–3-day-old wild-type, Glc-6-Pase-{alpha}–/–, and Glc-6-PT–/– pups. After 21 days in culture, nearly all of the cells from wild-type, Glc-6-Pase-{alpha}–/–, or Glc-6-PT–/– pups have the characteristics of astrocytes. Immunocytochemical analysis (Fig. 4A) shows that nearly all the cells stained positively for GFAP, a marker for brain astrocytes (32), and negatively for fibronectin and myelin basic protein, which are markers of fibroblasts and oligodendrocytes, respectively (31). Western blot analysis confirmed that the primary astrocytes from wild-type, Glc-6-Pase-{alpha}–/–, or Glc-6-PT–/– pups express GFAP (Fig. 4B) but not fibronectin or the myelin basic protein (data not shown). Importantly, primary astrocytes from wild-type, Glc-6-Pase-{alpha}–/–, and Glc-6-PT–/– pups express the Glc-6-Pase-{beta} protein in their microsomes (Fig. 4B), strongly suggesting that brain astrocytes may be capable of hydrolyzing Glc-6-P to glucose via a functional Glc-6-Pase-{beta} complex. To examine if this complex is functional, we assayed the astrocyte microsomes for Glc-6-P uptake and hydrolysis.



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 4.
Mouse astrocytes in primary culture express an active Glc-6-Pase-{beta}·Glc-6-PT complex. A, cell-specific immunostaining. Mouse astrocytes were isolated from 2–3-day-old wild-type, Glc-6-Pase-{alpha}–/–, or Glc-6-PT–/– pups and plated at a density of 9 x 104 cells/6-cm dish. After 21 days in culture, the cells were stained for GFAP (a marker for astrocytes), fibronectin (a marker for fibroblasts), or myelin basic protein (a marker for oligodendrocytes) as described under "Experimental Procedures." The data shown are for astrocytes obtained from wild-type mice at magnifications of x1 (upper panels) and x50 (lower panels). Similar results were obtained with primary astrocytes from Glc-6-Pase-{alpha}–/– and Glc-6-PT–/– pups. B, Western blot analysis. Glc-6-Pase-{beta} in microsomes isolated from primary astrocytes of wild-type, Glc-6-Pase-{alpha}–/–, or Glc-6-PT–/– mice were detected using anti-hGlc-6-Pase-{beta} (22). The lane marked mGlc-6-Pase-{beta} represents protein from Ad-mGlc-6-Pase-{beta}-infected COS-1 lysates. GFAP in cell lysates was detected using a rabbit anti-GFAP antibody. C, astrocyte phosphohydrolase and Glc-6-P uptake activities. Glc-6-P hydrolytic and uptake assays were performed using microsomes isolated from primary astrocytes of wild-type, Glc-6-Pase-{alpha}–/–, and Glc-6-PT–/– mice as described under "Experimental Procedures." The results are given as mean ± S.E.

 
The acid-sensitive Glc-6-P hydrolase activities in microsomes isolated from the astrocyte primary cultures of wild-type, Glc-6-Pase-{alpha}–/–,and Glc-6-PT–/– mice are similar, ranging from 15.0 to 15.7 nmol/mg/min (Fig. 4C) and accounting for ~7% of the activity of the liver (22). This is ~50% higher than the specific activities in microsomes isolated from the whole brain homogenates (Table I), suggesting, based on cell mass, that the astrocytes are the primary source of the brain activities.

A comparison of Glc-6-P uptake in primary astrocytes shows that Glc-6-P uptake activities in the intact microsomes of wild-type and Glc-6-Pase-{alpha}–/– mice are identical (Fig. 4C). In contrast, the Glc-6-PT–/– microsomes show a markedly reduced Glc-6-P accumulation (Fig. 4C), again consistent with the absence of Glc-6-PT in these mice (29).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The mammalian brain has no energy reserves, and a constant supply of oxygen and glucose is required for its function and survival. Blood glucose homeostasis between meals is maintained by balancing the blood glucose uptake by tissues with the release of Glc-6-P-derived glucose to the blood by the liver and kidney. Therefore, the dephosphorylation of Glc-6-P to glucose, catalyzed by the Glc-6-Pase complex in the terminal step of both gluconeogenesis and glycogenolysis, is a key control point for interprandial glucose homeostasis (1, 2). Until very recently, specific Glc-6-Pase activity has not been detected outside of the liver, kidney, and intestine. This has led to the current view that interprandial glucose homeostasis depends solely upon these organs. The recent recognition of Glc-6-Pase-{beta} (13, 14) and its universal expression profile (12, 33) has raised the possibility that glucose recycling might also occur to some extent in many tissues. The tissues that immediately became of interest to this hypothesis are those with substantial reserves of glycogen that could be converted to glucose via glycogenolysis and that also express the higher levels of Glc-6-Pase-{beta} and Glc-6-PT. Of particular interest are the skeletal muscle (21, 22), the brain, and the heart.

The Glc-6-Pase found in mouse brain in this study and the in vitro expressed mouse and human Glc-6-Pase-{beta} proteins have identical kinetic characteristics. All are acid-labile, readily inhibited by vanadate with [vanadate]0.5 of 1.3–1.4 mM, and all share optimal assay conditions of pH 6.5 and 37 °C, differing from those of Glc-6-Pase-{alpha} at pH 6.5 and 30 °C. These characteristics, along with the RNA and protein expression profiles, support the conclusion that the acid-labile Glc-6-P hydrolytic activity in brain is Glc-6-Pase-{beta}. Of particular significance, the brain Glc-6-Pase is capable of coupling to the ubiquitous Glc-6-PT to create a functional Glc-6-Pase complex capable of converting Glc-6-P to free glucose. In refining the site of this activity, we have shown that astrocytes, the main site of glycogen storage in the brain, contain a functional Glc-6-Pase-{beta}·Glc-6-PT complex in their microsomes, suggesting they are capable of hydrolyzing Glc-6-P to glucose and might be a source of endogenous brain glucose.

Whereas the Glc-6-P hydrolase activity in the brain (or astrocytes) represents only ~5–7% of the activity in the liver, the brain (or astrocyte) Glc-6-Pase-{beta}·Glc-6-PT complex has ~30–32% of the Glc-6-P transport activity of the liver Glc-6-Pase-{alpha}·Glc-6-PT complex. This is consistent with the previous reports that Glc-6-P translocation by Glc-6-PT is the rate-limiting step in Glc-6-Pase catalysis (19, 36). Our results indicate that despite the lower hydrolase activity in the brain, the overall activity of the Glc-6-Pase·Glc-6-PT complex in the brain is still sufficient to convert glycogen to glucose at a significant rate.

The potential role of brain glycogen in glucose homeostasis has been of significant interest. One concern that is expressed about the role of non-hepatic contributions to glucose homeostasis is based on kinetics. Glc-6-P is the intracellular form of extracellular glucose, trapped within the cell by the charged phosphate that prevents ready diffusion across the lipid cell membrane. The ability of the liver to maintain blood glucose homeostasis between meals is believed to stem, in part, from the balance between the Glc-6-P phosphohydrolase and glucose kinase activities present in the liver. The Glc-6-Pase-{alpha} activity in the liver is high, which allows for the rapid conversion of Glc-6-P derived from gluconeogenesis or glycogenolysis to glucose. Of the two glucose kinase activities, glucokinase and hexokinase, glucokinase predominates in the liver. Because glucokinase has the higher Km (~20 mM) (37, 38) compared with that of hexokinase (Km, ~50 µM) (24, 38), Glc-6-Pase-mediated glucose release to the blood can compete with rephosphorylation by glucokinase. On the other hand, most other tissues, including the kidney, express the low Km hexokinase. This might imply that in non-hepatic tissues, Glc-6-Pase-derived glucose is more likely to be rephosphorylated than released into the circulation. There is one clear inconsistency with this explanation, namely the observation that the kidney does play a proven role in blood glucose homeostasis, especially during prolonged starving (reviewed in Refs. 39 and 40). One possible explanation is that the kinetic reasoning does not consider the subcellular compartmentalization of the different activities. The fate of cytoplasmic glucose, exposed to the glucokinase and hexokinase activities, might be very different from that of glucose produced within the ER lumen by the Glc-6-Pase complex. Within the lumen, glucose is protected from the glucose kinase activities and may be released directly via the ER/Golgi pathway (2) where it is promptly taken up and re-phosphorylated to Glc-6-P by adjacent cells. Indeed, using GLUT2-deficient cells, it was demonstrated that hepatic (41) or intestinal (42) glucose release does not require the presence of the plasma membrane glucose facilitative diffusion mechanism, implying the existence of an alternative pathway for glucose release via a membrane traffic mechanism. This finding might explain why the cytoplasmic pathways of gluconeogenesis and glycogenolysis evolved a final step dependent upon a compartmentalized Glc-6-Pase enzyme complex.

The finding of a functional Glc-6-Pase complex in the brain astrocytes is consistent with a number of earlier observations and the known roles of astrocytes within the brain. It has been shown that astrocytes are the primary source of glycogen storage in the brain, containing sufficient glycogen reserves to buffer the majority of the glucose supply deficit for >100 min of hypoglycemia (24). It is also known that sensory stimulation causes a breakdown of astrocyte glycogen, whereas anesthesia and hibernation increase glycogen stores in the brain (35). These findings suggest a role of astrocyte glycogen metabolism and neuronal activity. The known roles of astrocytes, not only in structural support and regulation of the external chemical environment of the neurons but also in their roles directly supporting neuronal maturation and neurogenesis, would be consistent with an ability to regulate glucose release.

Although glucose export from the brain has been demonstrated in children undergoing elective cardiopulmonary bypass surgery for congenital heart disease (23), and we have shown here that astrocytes express an active Glc-6-Pase-{beta}·Glc-6-PT complex, there is very limited evidence that confers a glucose export capacity on astrocytes per se. Under most conditions in vitro, lactate export predominates. Primary cultures of astrocytes convert their glycogen into extracellular lactate upon glucose deprivation (38), and glycogen-derived lactate supports intact rodent optic nerve function in vitro during aglycemia and increased metabolic demand (43). Moreover, neuronal activation leads to an increase in lactate that can be used as a metabolic substrate by neurons, suggesting that energy is transferred from astrocytes to axons in the form of lactate (reviewed in Refs. 44 and 45).

Even though the proposal that astrocytes have the ability to convert glycogen to glucose can explain the source of glucose reported to be released from the brain under certain conditions of cardiopulmonary bypass (23), we do not know under what conditions the astrocyte glycogen is cycled into glucose in vivo. There are many fates of Glc-6-P in the cell, depending on the physiological status of the cell and its environment. These include lactate and/or energy production via glycolysis, entry into the pentose phosphate pathway, and glucose production via the Glc-6-Pase-{beta} complex in the ER. Because the level of glycogen storage in astrocytes is low compared with that in the liver and muscle, we do not consider glucose production to be a primary pathway in astrocytes but rather a pathway that may be used during periods of stress or metabolic restriction to buffer the brain function. The relative importance of the production and use of lactate and glucose in the brain remains a question for careful experimental scrutiny.

In conclusion, we have demonstrated that there is a functional Glc-6-Pase activity in the mouse brain that is present in astrocytes. The activity is independent of Glc-6-Pase-{alpha} and has the expected characteristics of Glc-6-Pase-{beta}. The activity can couple functionally with Glc-6-PT to produce an active Glc-6-Pase complex, and the complex can metabolize Glc-6-P to glucose and phosphate. These results can explain previous reports of the release of glucose from the brain and suggest that there is reason to think beyond simple lactate energy in the brain. The biological significance of this finding awaits more detailed studies.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Bldg. 10, Rm. 9S241, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1830. Tel.: 301-496-1094; Fax: 301-402-6035; E-mail: chouja{at}mail.nih.gov.

1 The 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. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chou, J. Y., Matern, D., Mansfield, B. C., and Chen, Y.-T. (2002) Curr. Mol. Med. 2, 121–143[CrossRef][Medline] [Order article via Infotrieve]
  2. Chen, Y.-T. (2001) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Childs, B., Kinzler, K. W., and Vogelstein, B., eds) 8th Ed., pp. 1521–1551, McGraw-Hill Inc., New York
  3. Hiraiwa, H., Pan, C.-J., Lin, B., Moses, S. W., and Chou, J. Y. (1999) J. Biol. Chem. 274, 5532–5536[Abstract/Free Full Text]
  4. Marcolongo, P., Barone, V., Priori, G., Pirola, B., Giglio, S., Biasucci, G., Zammarchi, E., Parenti, G., Burchell, A., Benedetti, A., and Sorrentino, V. (1998) FEBS Lett. 436, 247–250[CrossRef][Medline] [Order article via Infotrieve]
  5. Gerin, I., Veiga-da-Cunha, M., Noel, G., and Van Schaftingen, E. (1999) Gene 227, 189 –195[CrossRef][Medline] [Order article via Infotrieve]
  6. Lin, B., Pan, C.-J., and Chou, J. Y. (2000) Hum. Genet. 107, 526 –529[CrossRef][Medline] [Order article via Infotrieve]
  7. Lei, K.-J., Shelly, L. L., Pan, C.-J., Sidbury, J. B., and Chou, J. Y. (1993) Science 262, 580 –583[Abstract/Free Full Text]
  8. Shelly, L. L., Lei, K.-J., Pan, C.-J., Sakata, S. F., Ruppert, S., Schutz, G., and Chou, J. Y. (1993) J. Biol. Chem. 268, 21482–21485[Abstract/Free Full Text]
  9. Powell, R. C., Wentworth, S. M., and Brandt, I. K. (1981) Metabolism 30, 443–450[CrossRef][Medline] [Order article via Infotrieve]
  10. Tsalikian, E., Simmons, P., Gerich, J. E., Howard, C., and Haymond, M. W. (1984) Am. J. Physiol. 247, E513–E519[Medline] [Order article via Infotrieve]
  11. Collins, J. E., Bartlett, K., Leonard, J. V., and Aynsley-Green, A. (1990) J. Inherit. Metab. Dis. 13, 195–206[CrossRef][Medline] [Order article via Infotrieve]
  12. Martin, C. C., Oeser, J. K., Svitek, C. A., Hunter, S. I., Hutton, J. C., and O'Brien, R. M. (2002) J. Mol. Endocrinol. 29, 205–222[Abstract]
  13. Guionie, O., Clottes, E., Stafford, K., and Burchell, A. (2003) FEBS Lett. 551, 159 –164[CrossRef][Medline] [Order article via Infotrieve]
  14. Shieh, J.-J., Pan, C.-J., Mansfield, B. C., and Chou, J. Y. (2003) J. Biol. Chem. 278, 47098–47103[Abstract/Free Full Text]
  15. Pan, C.-J., Lei, K.-J., Annabi, B., Hemrika, W., and Chou, J. Y. (1998) J. Biol. Chem. 273, 6144–6148[Abstract/Free Full Text]
  16. Ghosh, A., Shieh, J.-J., Pan, C.-J., and Chou, J. Y. (2004) J. Biol. Chem. 279, 12479 –12483[Abstract/Free Full Text]
  17. Pan, C.-J., Lin, B., and Chou, J. Y. (1999) J. Biol. Chem. 274, 13865–13869[Abstract/Free Full Text]
  18. Pan, C.-J., Lei, K.-J., Chen, H., Ward, J. M., and Chou, J. Y. (1998) Arch. Biochem. Biophys. 358, 17–24[CrossRef][Medline] [Order article via Infotrieve]
  19. Nordlie, R. C., and Sukalski, K. A. (1985) in The Enzymes of Biological Membranes (Martonosi, A. N., ed) 2nd Ed., pp. 349 –398, Plenum Press, New York
  20. Ghosh, A., Shieh, J.-J., Pan, C.-J., Sun, M.-S., and Chou, J. Y. (2002) J. Biol. Chem. 277, 32837–32842[Abstract/Free Full Text]
  21. Baynes, J., and Dominiczak, M. H. (1999) in Medical Biochemistry (Baynes, J., ed) pp. 139–155, Mosby, London
  22. Shieh, J.-J., Pan, C.-J., Mansfield, B. C., and Chou, J. Y. (2004) J. Biol. Chem. 279, 26215–26219[Abstract/Free Full Text]
  23. Eyre, J. A., Stuart, A. G., Forsyth, R. J., Heaviside, D., and Bartlett, K. (1994) Brain Res. 635, 349 –352[Medline] [Order article via Infotrieve]
  24. Gruetter, R. (2003) J. Neurosci. Res. 74, 179 –183[CrossRef][Medline] [Order article via Infotrieve]
  25. Brown, A.M. (2004) J. Neurochem. 89, 537–552[CrossRef][Medline] [Order article via Infotrieve]
  26. Forsyth, R. J., Bartlett, K., Burchell, A., Scott, H. M., and Eyre, J. A. (1993) Biochem. J. 294, 145–151[Medline] [Order article via Infotrieve]
  27. Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y., and Phipps, M. L. (1997) J. Virol. 71, 1842–1849[Abstract]
  28. Lei, K.-J., Chen, H., Pan, C.-J., Ward, J. M., Mosinger, B., Lee, E. J., Westphal, H., and Chou, J. Y. (1996) Nat. Genet. 13, 203–209[CrossRef][Medline] [Order article via Infotrieve]
  29. Chen, 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., and Chou, J. Y. (2003) Hum. Mol. Genet. 12, 2547–2558[Abstract/Free Full Text]
  30. Burchell, A., Hume, R., and Burchell, B. (1988) Clin. Chim. Acta 173, 183–192[CrossRef][Medline] [Order article via Infotrieve]
  31. Pousset, F., Cremona, S., Dantzer, R., Kelley, K., and Parnet, P. (1999) Glia 26, 12–21[CrossRef][Medline] [Order article via Infotrieve]
  32. Rutka, J. T., Murakami, M., Dirks, P. B., Hubbard, S. L., Becker, L. E., Fukuyama, K., Jung, S., Tsugu, A., and Matsuzawa, K. (1997) J. Neurosurg. 87, 420–430[Medline] [Order article via Infotrieve]
  33. Boustead, J. N., Martin, C. C., Oeser, J. K., Svitek, C. A., Hunter, S. I., Hutton, J. C., and O'Brien, R. M. (2004) J. Mol. Endocrinol. 32, 33–53[Abstract]
  34. Hers, H. G. (1964) in Advances in Metabolic Disorders (Levine, R., and Luft, L., eds) Vol. 1, pp. 1–44, Academic Press, London
  35. Fillenz, M., Lowry, J. P., Boutelle, M. G., and Fray, A. E. (1999) Acta Physiol. Scand. 167, 275–284[CrossRef][Medline] [Order article via Infotrieve]
  36. Arion, W. J., Lange, A. J., Walls, H. E., and Ballas, L. M. (1980) J. Biol. Chem. 255, 10396 –10406[Abstract/Free Full Text]
  37. Parry, M. J., and Walker, D. G. (1966) Biochem. J. 99, 266 –274[Medline] [Order article via Infotrieve]
  38. Dringen, R., Gebhardt, R., and Hamprecht, B. (1993) Brain Res. 623, 208 –214[CrossRef][Medline] [Order article via Infotrieve]
  39. Gerich, J. E., Meyer, C., Woerle, H. J., and Stumvoll, M. (2001) Diabetes Care 24, 382–391[Abstract/Free Full Text]
  40. Cano, N. (2002) Critical Care 6, 317–321[CrossRef][Medline] [Order article via Infotrieve]
  41. Guillam, M. T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317–12321[Abstract/Free Full Text]
  42. Stumpel, F., Burcelin, R., Jungermann, K., and Thorens, B. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11330 –11335[Abstract/Free Full Text]
  43. Brown, A. M., Tekkok, S. B., and Ransom, B. R. (2004) Neurochem. Int. 45, 529 –536[CrossRef][Medline] [Order article via Infotrieve]
  44. Wiesinger, H., Hamprecht, B., and Dringen, R. (1997) Glia 21, 22–34[CrossRef][Medline] [Order article via Infotrieve]
  45. Pellerin L. (2003) Neurochem. Int. 43, 331–338[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
G. Oz, E. R. Seaquist, A. Kumar, A. B. Criego, L. E. Benedict, J. P. Rao, P.-G. Henry, P.-F. Van De Moortele, and R. Gruetter
Human brain glycogen content and metabolism: implications on its role in brain energy metabolism
Am J Physiol Endocrinol Metab, March 1, 2007; 292(3): E946 - E951.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Wang, J. K. Oeser, C. Yang, S. Sarkar, S. I. Hackl, A. H. Hasty, O. P. McGuinness, W. Paradee, J. C. Hutton, D. R. Powell, et al.
Deletion of the Gene Encoding the Ubiquitously Expressed Glucose-6-phosphatase Catalytic Subunit-related Protein (UGRP)/Glucose-6-phosphatase Catalytic Subunit-beta Results in Lowered Plasma Cholesterol and Elevated Glucagon
J. Biol. Chem., December 29, 2006; 281(52): 39982 - 39989.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11114    most recent
M410894200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed