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PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS
Lack of glucose recycling between endoplasmic reticulum and cytoplasm
underlies cellular dysfunction in glucose-6-phosphatase-␤–deficient neutrophils in
a congenital neutropenia syndrome
Hyun Sik Jun,1 Young Mok Lee,1 Yuk Yin Cheung,1 David H. McDermott,2 Philip M. Murphy,2 Suk See De Ravin,3
Brian C. Mansfield,1 and Janice Y. Chou1
1Section on Cellular Differentiation, Program on Developmental Endocrinology and Genetics, National Institute of Child Health and Human Development,
National Institutes of Health (NIH), Bethesda, MD; and 2Laboratory of Molecular Immunology and 3Laboratory of Host Defenses, National Institute of Allergy and
Infectious Diseases, NIH, Bethesda, MD
G6PC3 deficiency, characterized by neutropenia and neutrophil dysfunction, is caused
by deficiencies in the endoplasmic reticulum (ER) enzyme glucose-6-phosphatase-␤
(G6Pase-␤ or G6PC3) that converts glucose6-phosphate (G6P) into glucose, the primary
energy source of neutrophils. Enhanced
neutrophil ER stress and apoptosis underlie
neutropenia in G6PC3 deficiency, but the
exact functional role of G6Pase-␤ in neutrophils remains unknown. We hypothesized
that the ER recycles G6Pase-␤–generated
glucose to the cytoplasm, thus regulating
the amount of available cytoplasmic glucose/
G6P in neutrophils. Accordingly, a G6Pase-␤
deficiency would impair glycolysis and hexose monophosphate shunt activities leading to reductions in lactate production,
adenosine-5ⴕ-triphosphate (ATP) production, and reduced nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase
activity. Using annexin V–depleted neutrophils, we show that glucose transporter-1
translocation is impaired in neutrophils from
G6pc3ⴚ/ⴚ mice and G6PC3-deficient patients along with impaired glucose uptake in
G6pc3ⴚ/ⴚ neutrophils. Moreover, levels of
G6P, lactate, and ATP are markedly lower in
murine and human G6PC3-deficient neutrophils, compared with their respective controls. In parallel, the expression of NADPH
oxidase subunits and membrane translocation of p47phox are down-regulated in murine
and human G6PC3-deficient neutrophils.
The results establish that in nonapoptotic
neutrophils, G6Pase-␤ is essential for normal energy homeostasis. A G6Pase-␤ deficiency prevents recycling of ER glucose to
the cytoplasm, leading to neutrophil dysfunction. (Blood. 2010;116(15):2783-2792)
Introduction
There are 2 enzymatically active glucose-6-phosphatases (G6Pases),
the liver/kidney/intestine–restricted G6Pase-␣ (or G6PC)1,2 and the
ubiquitously expressed G6Pase-␤ (also known as G6PC3).3,4 Both
enzymes are transmembrane endoplasmic reticulum (ER) proteins,
with a similar topology, that places their active site on the luminal
side of the ER membrane.5,6 Both have similar kinetic properties2,4
and hydrolyze glucose-6-phosphate (G6P) to glucose and phosphate when coupled with the ubiquitously expressed G6P transporter (G6PT) that translocates G6P from the cytoplasm into the
lumen of the ER.7,8 The primary role of the G6Pase/G6PT complex
is to provide glucose and phosphate to the ER lumen. The
G6Pase-␣/G6PT complex maintains blood glucose homeostasis
between meals by hydrolyzing G6P to glucose in the terminal step
of gluconeogenesis and glycogenolysis.1,2 Deficiencies in G6Pase-␣
cause glycogen storage disease type Ia (GSD-Ia) and deficiencies in
G6PT result in GSD type Ib (GSD-Ib).1,2,9 Both GSD-Ia and
GSD-Ib patients manifest a phenotype of disturbed blood glucose
homeostasis with GSD-Ib patients also suffering neutropenia and
neutrophil dysfunction,1,9 reflecting a role of G6PT in tissues
beyond the liver and kidney.
The biologic roles of G6Pase-␤ and the G6Pase-␤/G6PT
complex are poorly defined. Neutrophils, which express both
G6Pase-␤ and G6PT,10 are capable of endogenous production of
glucose, the primary energy for neutrophils,11 which might suggest
that G6Pase-␤ and G6PT play a role in neutrophil homeostasis and
function. To examine this hypothesis, we generated mouse lines
deficient in either G6Pase-␤10 or G6PT12 and showed that both
G6Pase-␤–deficient (G6pc3⫺/⫺) and G6PT-deficient (GSD-Ib) mice
manifest neutropenia and neutrophil dysfunction that mimic the
symptoms in human GSD-Ib patients.1,9 We further showed that
neutrophils deficient in either G6Pase-␤10 or G6PT13 undergo
enhanced ER stress and apoptosis, demonstrating that endogenous
glucose production in the ER is critical for neutrophil homeostasis.
This is consistent with the observation that human GSD-Ib patients
manifest a phenotype of congenital neutropenia.1,9 Providing
further support for the vital role of G6Pase-␤/G6PT in normal
neutrophil function, a recent linkage analysis identified the G6PC3
gene as the disease candidate for a severe congenital neutropenia
syndrome.14 Moreover, neutrophils from G6PC3-deficient patients
also exhibited enhanced ER stress and apoptosis.14
Neutrophils play a key role in the early inflammatory response
to infection. Because neutrophils cannot produce glucose via
gluconeogenesis,15 their glucose is supplied either by the breakdown of stored glycogen, or by uptake from the blood via
facilitated diffusion across the plasma membrane mediated by the
glucose transporters (GLUTs).16 Within the neutrophil, glucose is
Submitted December 21, 2009; accepted May 12, 2010. Prepublished online as
Blood First Edition paper, May 24, 2010; DOI 10.1182/blood-2009-12-258491.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
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BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
JUN et al
metabolized by hexokinase to G6P.11 There are multiple competing
cytoplasmic pathways that use intracellular glucose/G6P, including
glycogen formation,11,17 glycolysis,11,18 the hexose monophosphate
shunt (HMS),11,19 and G6PT-mediated ER uptake. Glycogen use in
neutrophils is considered primarily restricted to phagocytic activity.20 Previous views held that the primary competing glucose/G6P
pathways in neutrophils are glycolysis and HMS. Glycolysis
supplies the necessary energy for neutrophil locomotion and
chemotaxis.11,18 HMS generates the reduced nicotinamide adenine
dinucleotide phosphate (NADPH) substrate for NADPH oxidase,
which facilitates the production of superoxide anions and hence
other reactive oxygen species (ROS).21,22 However, more recent
studies show that G6P transported into the ER is metabolized by at
least 2 different enzymes: G6Pase-␤ to produce glucose4,5 and
hexose-6-phosphate dehydrogenase (H6PDH) to generate NADPH
that is important for in vivo function of 11␤-hydroxysteroid
dehydrogenase-1.23 H6PDH-deficient mice are not reported to have
neutrophil abnormalities.24,25 In contrast, G6Pase-␤–deficient
mice do manifest neutrophil dysfunction10 that mimic those in
GSD-Ib patients.1,9 This suggests that G6Pase-␤ provides the
primary pathway of glucose metabolism in the ER and represents an additional G6P-utilization pathway that regulates
neutrophil function.
We have demonstrated that endogenous glucose production in the
ER via the G6Pase-␤/ G6PT complex is critical for normal neutrophil
function10 but the exact role and mechanism of action of G6Pase-␤ in
phagocytes remains to be elucidated. We now propose that ER recycling
of G6P is a third major competing pathway regulating cytoplasmic
glucose/G6P in neutrophils. We hypothesize that ER uptake of G6P by
the G6PT provides substrate to G6Pase-␤, which then releases glucose
back to the cytoplasm, as a rapid and responsive mechanism for
regulating the amount of available cytoplasmic glucose/G6P. Accordingly, we predict that neutrophils lacking G6Pase-␤ will be unable to
release glucose from the ER into the cytoplasm. As a result, cytoplasmic
glucose/G6P levels in G6Pase-␤–deficient neutrophils will fall, if not
countered by an appropriate increase in blood glucose uptake. Such
falling levels of intracellular G6P are predicted to impair glycolysis and
HMS activities, which will lead to decreases in neutrophil lactate and
adenosine-5⬘-triphophate (ATP) levels and neutrophil dysfunction charcterized by impaired respiratory burst, chemotaxis, calcium flux,
and phagocytosis.
In this report we present evidence consistent with this hypothesis. Because G6PC3 deficiency is characterized by neutropenia
and enhanced neutrophil apoptosis,10,14 functional assessments
were conducted in neutrophils purified by annexin V depletion of
apoptotic cells. We show that the intracellular levels of G6P,
lactate, and ATP in neutrophils of G6pc3⫺/⫺ mice and human
G6PC3-deficient patients are markedly reduced compared with the
controls. Likewise, NADPH oxidase expression and activation are
impaired in both human and murine G6PC3-deficient neutrophils.
In normal neutrophils, increasing cytoplasmic lactate and ATP
levels stimulate uptake of blood glucose by inducing the translocation of GLUT1 to the plasma membrane.26-28 Consistent with this
we show that the GLUT1 translocation to the plasma membrane is
impaired in human and murine G6PC3-deficient neutrophils.
Moreover, glucose uptake is impaired in G6pc3⫺/⫺ neutrophils.
Taken together, our results support the hypothesis that the underlying cause of neutrophil dysfunction in G6PC3 deficiency is a
disturbance in ER energy homeostasis caused by a loss of the
endogenous glucose/G6P reservoir in neutrophils.
Methods
Isolation of murine bone marrow and human blood neutrophils
Animal studies were conducted under a protocol approved by the National
Institute of Child Health and Human Development Animal Care and Use
Committee. The 2 G6PC3-deficient patients, both under granulocytecolony stimulating factor (G-CSF) therapy, were studied, after written
informed consent was received, under a National Institute of Allergy and
Infectious Diseases Institutional Review Board. The congenic G6pc3⫺/⫺
mice were obtained by backcrossing G6pc3⫺/⫺ mice10 to C57BL/6J mice
for 12 generations. Bone marrow (BM) cells were isolated from the femurs
and tibiae of 6- to 8-week-old G6pc3⫺/⫺ and control littermates, and
neutrophils were purified from the BM cells using the EasySep negative
selection system (StemCell Technologies). The antibody selection cocktail contained CD5/CD4/CD45R for thymocytes, T and B lymphocytes;
TER119 for erythroid cells; and F4/80 for monocytes/macrophages. The
purity of BM neutrophils was monitored by Wright staining and confirmed
by immunocytochemistry using a rat monoclonal antibody against Gr-1
(BD Pharmingen). Neutrophil viability was determined by trypan blue
staining and the viable neutrophils were counted using the Countess
Automated Cell Counter (Invitrogen).
Human peripheral blood neutrophils were isolated from heparinized
blood using EasySep human neutrophil enrichment kit (StemCell Technologies). The antibody cocktail is composed of CD2, CD3, CD9, CD19, CD36,
CD56, glycophorin A, and dextran, to remove lymphocytes, monocytes,
erythrocyte, platelets, NK, and dendritic cells. The purity of human
neutrophils was monitored by Wright staining.
Annexin V binding and caspase-3 activity assays
Neutrophil apoptosis was assessed using the annexin V–FITC apoptosis
detection kit (BioVision) and analyzed by flow cytometry as described
previously,13 using the Guava EasyCyte Mini System (Millipore). Caspase-3
activity in neutrophil lysates was measured using labeled Asp-Glu-Val-Aspp-nitroanilide (DEVD-pNA) and the caspase-3 colorimetric assay kit
(BioVision) as described previously.10
Depletion of apoptotic cells from neutrophils
The Annexin V Microbead kit (Miltenyi Biotec) was used to deplete
apoptotic cells in the isolated neutrophils. Briefly, freshly isolated control
and G6PC3-deficient neutrophils were incubated with Annexin V Microbeads for 15 minutes at 6°C and the mixture passed through a MACS
cell separation column (Miltenyi Biotec). The annexin V-negative cells
were pelleted, resuspended in the appropriate buffer, and used for all assays
except ER stress and apoptosis.
Respiratory burst, chemotaxis, calcium flux and phagocytosis
measurements
Respiratory burst, chemotaxis, and calcium flux analysis were performed as
previously described.10,12
The phagocytic activity of neutrophils was examined by measuring the
uptake of pHrodo Escherichia coli bioparticles (Invitrogen) as described by
Wan et al29 The number of neutrophils with phagocytosed bioparticles was
visualized using the Axioskop2 plus fluorescence microscope, quantified by
Adobe Photoshop CS3 (Adobe Systems), and expressed as [(number of
cells with phagocytosed particles)/(total cell numbers analyzed) ⫻ 100].
Glucose uptake
Glucose was measured by the rate of 2-deoxy-D-[1,2-3H]-glucose (2-DG)
uptake as described by Bashan and coworkers.30 Briefly, annexin V–
depleted BM neutrophils (2 ⫻ 106) were incubated at 37°C for 2 to
10 minutes in 1 mL of Kreb’s Ringer phosphate (KRP) buffer containing
5 ␮Ci of 2-DG (33 Ci/mmol; MP Biomedicals). The reaction was stopped
with 1 mL of ice-cold KRP buffer and centrifuged. The washed pellets were
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BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
lysed in 0.5 mL of 1% (vol/vol) Triton-X100 in phosphate-buffered saline
(PBS) and protein content and radioactivity in cell lysates were determined.
Quantitative real-time RT-PCR and Western blot analyses
NEUTROPHIL DYSFUNCTION IN G6PC3 DEFICIENCY
2785
590 nm using a Flexstation II Fluorimeter. A standard curve was generated
by measuring released glucose after hydrolysis of various amounts of
glycogen (rabbit liver, type III; Sigma-Aldrich) by amyloglucosidase under
the same conditions.
Total RNAs were isolated from annexin V–depleted neutrophils using the
TRIzol Reagent (Invitrogen). The mRNA expression was quantified by
real-time reverse-transcription–polymerase chain reaction (RT-PCR) in an
Applied Biosystems 7300 Real-Time PCR System. The following TagMan
probes were used: gp91phox, Mm00514478_m1; p22phox, Mm00514478_m1;
p47phox, Mm00447921_m1; Glut1, Mm00441480_m1; and ␤-actin,
Mm00607939_s1. Data were analyzed using SDS Version 1.3 software
(Applied Biosystems) and normalized to ␤-actin RNA.
Western blot analysis was performed as previously described.10,12 The
following antibodies used were: a goat polyclonal antibody against
gelatinase (or MMP-9; Santa Cruz Biotechnologies), a mouse monoclonal
antibody against KDEL (Assay Designs), protein disulfide isomerase
(PDI; Novus Biologicals), or gp91phox (BD Biosciences); a rabbit polyclonal antibody against GLUT1, p22phox, or p47phox (Santa Cruz Biotechnologies), or GRP17031; a mouse polyclonal antibody against ␤-actin (Santa
Cruz Biotechnologies), or a rat monoclonal antibody against Gr-1
(BD Pharmingen).
Total ATP determination
Immunofluorescence microscopic analyses
Results
Mouse BM neutrophils or human blood neutrophils were deposited on glass
slides by cytospin, fixed for 10 minutes in 3.75% formaldehyde, permeabilized for 10 minutes at 25°C in 0.2% Triton X-100, incubated with Image-iT
FX signal enhancer (Invitrogen) for 30 minutes at 25°C, then blocked for
1 hour at 25°C in PBS containing 5% goat serum. The permeabilized
neutrophils were incubated for 1 hour at 25°C with a mouse polyclonal
antibody against gp91phox or pan Cadherin (Abcam), a rabbit polyclonal
antibody against GLUT1, p22phox, or p47phox in PBS supplemented with 5%
goat serum. After washes with PBS, neutrophils were incubated for 1 hour
at 25°C in the dark with an appropriate secondary antibody and conjugated
with either Alexa Fluor 488 or 555 (Invitrogen). The labeled cells were
mounted with an antifade, water-based mounting medium containing
4⬘,6-diamidino-2-phenylindole (DAPI) and visualized using a Zeiss Axiovert 200M inverted confocal microscope equipped with 40⫻/1.3 numeric
aperture (NA) or 63⫻/1.4 NA oil objectives at room temperature (Carl
Zeiss MicroImaging). The images were acquired using LSM 5 acquisition
software (Carl Zeiss Microimaging).
G6pc3ⴚ/ⴚ bone marrow is neutropenic and exhibits neutrophil
ER stress and apoptosis
G6P, lactate, and glycogen determination
Annexin V–depleted neutrophils were incubated for 30 minutes at 37°C in
5.6mM glucose-containing RPMI-1640 medium. For intracellular G6P,
lactate, and glycogen determination, 107 neutrophils in 500 ␮L of ice-cold
PBS were deproteinized on ice for 10 minutes with 125 ␮L of perchloric
acid (14%, wt/vol). After removing the denatured proteins by centrifugation, the acid supernatants (500 ␮L) were neutralized with 133 ␮L of 2M
KOH/0.2M MOPS, incubated on ice for 5 minutes, and precipitates
removed by centrifugation. The supernatants were stored at ⫺80°C.
To measure G6P, 10 ␮L of deproteinized neutrophil lysate was
incubated for 1 hour at 25°C in a 96-well microplate (Greiner Bio-One) in
100 uL of Hanks buffered salt solution containing 1 unit of yeast G6P
dehydrogenase (G6PDH; Sigma-Aldrich) and 4mM NADP⫹ (SigmaAldrich). Oxidation of G6P to 6-phosphogluconlactone reduced NADP⫹ to
NADPH, which was measured by excitation at 340 nm and emission at
450 nm32 in a Flexstation II Fluorimeter.
Lactate in deproteinized neutrophil lysates was analyzed using a kit
obtained from BioVision and the fluorescence intensities were measured by
excitation at 535 nm and emission at 575 nm using a Flexstation II
Fluorimeter.
Glycogen in the deproteinized neutrophil lysates was measured as
released glucose, using a glucose assay kit obtained from BioVision, after
hydrolysis in 0.1M sodium acetate buffer, pH 4.5 containing 1 mg/mL
amyloglucosidase (38 U/mg; Sigma-Aldrich) for 1 hour at 55°C. Fluorescence intensities were measured by excitation at 530 nm and emission at
Annexin V–depleted neutrophils (5 ⫻ 105) were incubated in 5.6mM
glucose-containing RPMI-1640 medium at 37°C for 30 minutes. Total ATP
was determined using the ATP fluorometric assay kit (BioVision). Briefly,
neutrophils were pelleted, lysed in 50 ␮L of ATP assay buffer, and
centrifuged. The supernatant (5 ␮L) was mixed with ATP Probe, ATP
Converter, and Developer Mix and incubated for 30 minutes at room
temperature in the dark. The fluorescence intensity was determined by
excitation at 535 nm and emission at 587 nm using a Flexstation II
Fluorimeter.
Statistical analysis
The unpaired t test was performed using the GraphPad Prism Program,
Version 4 (GraphPad Software). Values were considered statistically
significant at P less than .05.
Previous studies have shown that G6pc3⫺/⫺ mice are neutropenic,
with differential peripheral blood neutrophil counts in 6- to
7-week-old G6pc3⫺/⫺ mice averaging 67% of the counts in
age-matched unaffected littermates.10 We examined neutrophil
counts in total BM cells, isolated from the femur and tibia of 6- to
8-week-old G6pc3⫺/⫺ and control (G6pc3⫹/⫺/G6pc3⫹/⫹) littermates. The total cells recovered in the BM aspirates for G6pc3⫺/⫺
mice (4.2 ⫻ 107 ⫾ 0.04 ⫻ 107 cells) were, on average, 85% of
those in control mice (5.0 ⫻ 107 ⫾ 0.09 ⫻ 107 cells). Morphologically mature neutrophils, including band and segmented nucleophilic cells that were more than 95% pure, were isolated from
G6pc3⫺/⫺ and control mice (Figure 1A) by adopting a negative
immunomagnetic system.33 Both populations expressed similar
levels of Gr-1 (Figure 1B), a marker of neutrophil maturation,34 and
gelatinase (Figure 1B), a marker of terminal neutrophil differentiation,35 indicating that the majority of G6pc3⫺/⫺ and control BM
neutrophils were of similar biochemical maturity. Flow cytometric
analysis using anti–Gr-1 and anti-CD11b antibodies showed that
neutrophils in G6pc3⫺/⫺ BM averaged only 58.5% of the counts in
the BM of age-matched control littermates (Figure 1C). Therefore,
neutropenia in G6pc3⫺/⫺ mice is present within the BM before
extravasation.
We have previously shown that thioglycollate-stimulated peritoneal neutrophils from G6pc3⫺/⫺ mice exhibit enhanced ER stress
and apoptosis.10 We therefore examined the expression of molecular chaperones in the unfolded protein response signal transduction
pathway between BM neutrophils of G6pc3⫺/⫺ and control mice.
Western blot analysis showed that the production of molecular
chaperones, GRP78/Bip,36 GRP170,36 and PDI,36 were increased in
BM neutrophils of G6pc3⫺/⫺ mice relative to their control littermates (Figure 1D), implying that resting G6pc3⫺/⫺ neutrophils also
undergo enhanced ER stress.
Flow cytometric analysis showed that significantly elevated
numbers of BM neutrophils from G6pc3⫺/⫺ mice stained positive
for annexin V37 compared with neutrophils from the control mice
(Figure 1E), suggesting there was an enhanced rate of apoptosis in
G6pc3⫺/⫺ BM neutrophils. Consistent with this, activity assays
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JUN et al
BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
Figure 1. G6pc3ⴚ/ⴚ BM neutrophils display enhanced ER stress
and apoptosis. Bone marrow (BM) neutrophils were isolated from
6- to 8-week-old unaffected (⫹/⫹) and G6pc3⫺/⫺ (⫺/⫺) littermates
as described in “Isolation of murine bone marrow and human blood
neutrophils.” (A) Hema 3–stained cytospins of BM neutrophils.
(B) Western blot analysis of protein extracts of neutrophils using
antibodies against Gr-1, gelatinase, or ␤-actin. Data from 2 pairs of
littermates are shown and each lane contains 80 ␮g of protein.
(C) A plot of BM neutrophil counts in control and G6pc3⫺/⫺ mice
determined by flow cytometry analysis using anti–Gr-1 and antiCD11b antibodies (n ⫽ 4). (D) Western blot analysis of protein
extracts of neutrophils using antibodies against GRP78, GRP170,
PDI, or ␤-actin. Data from 2 pairs of littermates are shown and each
lane contains 50 ␮g of protein. (E) Quantification of apoptotic cells
(annexin V⫹) in BM neutrophils of control and G6pc3⫺/⫺ mice
determined by flow cytometric analysis. At least 5000 cells were
used for each determination (n ⫽ 4). (F) The DEVD-cleaving activity
of active caspase-3 in protein extracts of BM neutrophils. Data
represent the mean ⫾ SEM of 3 independent experiments. *P ⬍ .05,
**P ⬍ .005.
demonstrated that levels of active caspase-338 were increased in
G6pc3⫺/⫺ BM neutrophils (Figure 1F).
In summary, both peritoneal10 and BM neutrophils from G6pc3⫺/⫺
mice as well as peripheral blood neutrophils of G6PC3-deficient patients14 exhibit enhanced apoptosis, which underlies neutropenia in
G6PC3 deficiency. Therefore, to measure neutrophil function accurately, we removed apoptotic cells from purified BM neutrophils by
annexin V binding. The annexin V–depleted neutrophils from control
and G6pc3⫺/⫺ mice were of similar viability (Figure 2A). Moreover,
cell viabilities were unchanged after incubation in 5.6mM glucose for
30 minutes (Figure 2A). The experiments described in the remaining
subsections were all conducted in annexin V–depleted BM neutrophils.
G6pc3ⴚ/ⴚ murine bone marrow neutrophils exhibit
impaired function
We have previously shown that thioglycollate-stimulated peritoneal neutrophils from G6pc3⫺/⫺ mice exhibit impaired respiratory
Figure 2. Annexin V–depleted BM neutrophils from G6pc3ⴚ/ⴚ mice exhibit impaired function. The annexin V–depleted BM neutrophils were isolated from 6- to
8-week-old unaffected (⫹/⫹, E) and G6pc3⫺/⫺ (⫺/⫺, F) littermates as described in “Depletion of apoptotic cells from neutrophils.” (A) Neutrophil viability. The viability of
annexin V–depleted BM neutrophils before (Glucose ⫺) or after incubation for 30 minutes in 5.6mM glucose (Glucose ⫹) was estimated by trypan blue exclusion. Results
represent the mean ⫾ SEM of quadruplet determinations. (B) Neutrophil respiratory burst activity in response to 200 ng/mL PMA. (C) Neutrophil concentration–dependent
chemotaxis in response to fMLP and KC. *P ⬍ .05. (D) Calcium flux in response to 10⫺6M fMLP or KC. Representative experiments are shown. (E) Neutrophil phagocytosis
activity. Representative immunofluorescence of cells with phagocytosed pHrodo E coli bioparticles (red fluorescence) and DAPI nuclei staining (blue fluorescence) at 400⫻
magnification, and quantification of bioparticle-positive neutrophils incontrol and G6pc3⫺/⫺ mice. Data represent the mean ⫾ SEM of 3 independent experiments. **P ⬍ .005.
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BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
NEUTROPHIL DYSFUNCTION IN G6PC3 DEFICIENCY
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Figure 3. Analysis of 2-DG uptake, the expression of GLUT1, and
intracellular G6P, lactate, ATP, and glycogen levels in G6pc3ⴚ/ⴚ
neutrophils. Annexin V–depleted BM neutrophils were isolated from
6- to 8-week-old unaffected (⫹/⫹) and G6pc3⫺/⫺ (⫺/⫺) littermates as
described in “Depletion of apoptotic cells from neutrophils.” Freshly
isolated annexin V–depleted neutrophils were used for 2-DG uptake,
quantitative RT-PCR, and Western blot analyses. Immunofluorescence analysis of GLUT1 and measurement of G6P, lactate, ATP, and
glycogen were conducted in annexin V–depleted neutrophils that
were incubated for 30 minutes at 37°C in glucose-containing RPMI1640 medium as described in “Immunofluorescene microscopic analyses”; “G6P, lactate, and glycogen determination”; and “Total ATP
determination.” For 2-DG uptake and quantitative RT-PCR, the data
represent the mean ⫾ SEM of 3 independent experiments. **P ⬍ .005.
For Western blot and immunofluorescence analysis, at least 3 separate experiments were conducted in which each mouse was assessed
individually. (A) Uptake of 2-DG. (B) Quantification of GLUT1 mRNA
by real-time RT-PCR. (C) Western blot analysis of protein extracts of
annexin V–depleted BM neutrophils using antibodies against GLUT1
or ␤-actin. Each lane contains 50 ␮g of protein. The relative GlUT1
protein levels were quantified by densitometry of 4 separate pairs of
Western blots. *P ⬍ .05. (D) Representative immunofluorescence of
GLUT1 staining (green fluorescence), pan Cadherin membrane staining (red fluorescence), and DAPI nuclei staining (blue fluorescence) at
400⫻ magnification. (E) G6P, lactate, ATP, and glycogen levels. Data
represent the mean ⫾ SEM of 4 independent experiments. *P ⬍ .05.
burst, chemotaxis, and Ca2⫹ flux activities.10 We examined neutrophil function in resting BM neutrophils purified by annexin V
depletion of apoptotic cells. In control BM neutrophils, superoxide
production was markedly increased by exposure to phorbol 12myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) while in G6pc3⫺/⫺ BM neutrophils, the PMA- or
fMLP-stimulated superoxide production was reduced (Figure 2B).
BM neutrophils from unaffected mice exhibited a greater dosedependent chemotactic response to fMLP and keratinocyte chemoattractant (KC) than BM neutrophils from G6pc3⫺/⫺ mice (Figure
2C). Similarly, mobilization of calcium in response to fMLP and
KC was impaired in G6pc3⫺/⫺ BM neutrophils relative to controls
(Figure 2D). Therefore, BM and peritoneal neutrophils from
G6pc3⫺/⫺ mice behave in a similar manner, both exhibiting
impaired respiratory burst, chemotaxis, and calcium flux activities.
The phagocytic activity of nonapoptotic G6pc3⫺/⫺ and control
BM neutrophils was examined by measuring the uptake of pHrodo
E coli bioparticles.29 In control mice, 30.5% (⫾ 1.8%) of BM
neutrophils ingested the bioparticles (Figure 2E). In contrast in
G6pc3⫺/⫺ mice only 15.2% (⫾ 1.1%) of BM neutrophils harbored
ingested bioparticles (Figure 2E), consistent with an impairment in
phagocytosis.
ATP, which in turn will lead to impaired GLUT1 membrane
translocation and reduced glucose uptake, the rate-limiting step in
glucose metabolism.39 The nonapoptotic neutrophils from unaffected mice took up 2-DG at a rate 1.6-fold greater than neutrophils
from G6pc3⫺/⫺ mice (Figure 3A). As expected, the higher level of
glucose uptake by BM neutrophils from control mice was associated with higher levels of GLUT1 expression and increased
GLUT1 membrane-association. Quantitative real-time RT-PCR
analysis showed that neutrophil GLUT1 mRNA levels in G6pc3⫺/⫺
mice are, on average, 31% of the levels in their age-matched
control littermates (Figure 3B). Western blot analysis confirmed the
reduction in GLUT1 protein (Figure 3C). Immunofluorescence
analysis using an antibody to GLUT1 showed that the expression of
membrane associated GLUT1 was markedly decreased in G6pc3⫺/⫺
neutrophils, compared with the controls (Figure 3D). Glucose
uptake can also be increased by translocation of hexokinase to the
plasma membrane.40 We observed no difference in membrane
association of hexokinase between control and G6pc3⫺/⫺ neutrophils (data not shown).
Consistent with these findings, intracellular G6P levels in BM
neutrophils from G6pc3⫺/⫺ mice were only 34.8% of the control
mice (Figure 3E).
Impaired glucose uptake and reduced G6P levels in G6pc3ⴚ/ⴚ
neutrophils
Reduced levels of lactate and ATP in G6pc3ⴚ/ⴚ neutrophils
We hypothesized that the role of G6Pase-␤ is to cycle and regulate
the amount of available cytoplasmic glucose/G6P. We predict that
G6pc3⫺/⫺ neutrophils will have lower levels of G6P, lactate, and
Glycolysis, a primary source of energy in the neutrophils, generates
ATP by converting G6P to lactate.11,18 Therefore, a reduction in
available glucose/G6P is expected to impair glycolysis. We showed
that lactate levels in G6pc3⫺/⫺ neutrophils were 37% of the levels
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JUN et al
BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
Figure 4. Analysis of the expression of NADPH oxidase in G6pc3ⴚ/ⴚ neutrophils. Annexin V–depleted BM neutrophils were isolated from 6- to 8-week-old unaffected
(⫹/⫹) and G6pc3⫺/⫺ (⫺/⫺) littermates as described in “Depletion of apoptotic cells from neutrophils.” (A) Quantification of gp91phox, p22phox, and p47phox mRNA by real-time
RT-PCR. Data represent the mean ⫾ SEM of 3 independent experiments. *P ⬍ .05, **P ⬍ .005. (B) Representative immunofluorescence analysis of gp91phox (red
fluorescence) or p22phox (green fluorescence) and DAPI nuclei staining (blue fluorescence) at 400⫻ magnification. (C) Western blot analysis of protein extracts using
antibodies against gp91phox, p22phox, p47phox or ␤-actin. Data from 2 pairs of littermates are shown and each lane contains 50 ␮g of protein. The relative protein levels of
gp91phox, p22phox, p47phox were quantified by densitometry of 4 separate pairs of Western blots. *P ⬍ .05; **P ⬍ .005. (D) Representative immunofluorescence analysis of
p47phox (green fluorescence), pan Cadherin membrane staining (red fluorescence), and DAPI nuclei staining (blue fluorescence) at 400⫻ magnification.
in control neutrophils (Figure 3E). These results were supported by
measurements of total cellular ATP. In G6pc3⫺/⫺ mice, neutrophil
ATP levels were 69% of the levels in control mice (Figure 3E). We
also examined glycogen levels and showed that glycogen levels
were similar between control and G6pc3⫺/⫺ BM neutrophils
incubated both in the presence (Figure 3E) or absence of glucose
(data not shown).
In summary, G6pc3⫺/⫺ neutrophils exhibited impaired glucose
uptake and harbored reduced intracellular levels of G6P, lactate,
and ATP compared with control neutrophils.
Impaired expression and activation of NADPH oxidase in
G6pc3ⴚ/ⴚ neutrophils
NADPH oxidase is a multicomponent enzyme system composed of
2 transmembrane proteins, gp91phox and p22phox, and several
cytosolic proteins.21,22 The enzyme derives its substrate, NADPH,
from the HMS pathway.11 We examined the expression of gp91phox
and p22phox in nonapoptotic BM neutrophils from G6pc3⫺/⫺ and
unaffected littermates. Quantitative real-time RT-PCR analysis
showed that gp91phox mRNA levels in neutrophils of G6pc3⫺/⫺
mice were, on average, 55.5% of the levels in neutrophils of their
age-matched control littermates (Figure 4A).
Immunofluorescence analysis using an antibody to gp91phox
showed that the expression of membrane associated gp91phox was
also markedly decreased in G6pc3⫺/⫺ neutrophils, compared with
the controls (Figure 4B). In parallel, quantitative real-time RT-PCR
analysis showed that p22phox mRNA levels (Figure 4A) and the
expression of the membrane associated p22phox subunit (Figure 4B)
were also down-regulated in G6pc3⫺/⫺ neutrophils. Western blot
analysis confirmed the reduction in gp91phox and p22phox (Figure
4C), consistent with quantitative real-time RT-PCR and immunofluorescence results.
Activation of NADPH oxidase requires the translocation of
the p47phox subunit to the plasma membrane.41 Quantitative
real-time RT-PCR (Figure 4A) and Western blot (Figure 4C)
analyses showed that the expression of p47phox was decreased in
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BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
NEUTROPHIL DYSFUNCTION IN G6PC3 DEFICIENCY
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Figure 5. Analysis of levels of G6P, lactate, and ATP in neutrophils of human
G6PC3-deficient patients. Annexin V–
depleted blood neutrophils were isolated
from HD (n ⫽ 5; ages 23 to 37 years) and
2 G6PC3-deficient patients, P1 and P2, incubated for 30 minutes at 37°C in glucosecontaining RPMI-1640 medium, and G6P,
lactate, and ATP were determined as described in “G6P, lactate, and glycogen determination” and “Total ATP determination.”
(A) Hema 3-stained cytospins of neutrophils.
(B) G6P, lactate, and ATP levels. Data represent the mean ⫾ SEM of 2 independent
experiments, each done in duplicates.
*P ⬍ .05; **P ⬍ .005. (C) Representative immunofluorescence of GLUT1 staining (green
fluorescence), pan Cadherin membrane
staining (red fluorescence), and DAPI nuclei
staining (blue fluorescence) at 630⫻
magnification.
G6pc3⫺/⫺ neutrophils, compared with the controls. In the
absence of agonists, immunofluorescence analysis showed that
the p47phox subunit was primarily localized in the cytosol in
control and G6pc3⫺/⫺ neutrophils (Figure 4D), consistent with a
resting state of NADPH oxidase. In control neutrophils, PMA
activation induced p47phox translocation to the plasma membrane evidenced by the colocalization of p47phox with the plasma
membrane marker pan Cadherin (Figure 4D). In contrast, in
G6pc3⫺/⫺ neutrophils, the majority of p47phox remained in the
cytosol in the presence of PMA (Figure 4D). Therefore, the
impaired respiratory burst inherent of the G6pc3⫺/⫺ neutrophils
is associated with impairment of both the expression and
activation of NADPH oxidase.
Reduced levels of G6P, lactate, ATP, GLUT1, and NADPH
oxidase in human G6PC3-deficient neutrophils
We recently described 2 cases of G6PC3 deficiency, a 13-year-old
boy (P1) and his 9-year-old sister (P2), who both harbor the
homozygous G6PC3 mutation G260R.41 If the role of G6Pase-␤ is
to cycle and regulate the amount of available cytoplasmic glucose/
G6P, neutrophils from human G6PC3-deficient patients should also
exhibit impaired glycolysis. Morphologically mature neutrophils
from control health donors (HD) and G6PC3-deficient patients
(Figure 5A) were isolated by a negative immunomagnetic selection
system and purified by annexin V depletion. The intracellular G6P
levels in nonapoptotic neutrophils of P1 and P2 were on average
43.2% and 38.3%, respectively, of the levels in the control subjects
(Figure 5B). In parallel, intracellular lactate levels in P1 and P2
were 39.6% and 56.6%, respectively of the HD levels (Figure 5B).
Likewise, cellular ATP levels in P1 and P2 were 67.9% and 76.3%,
respectively, of the levels in the control subjects (Figure 5B). Taken
together, human G6PC3-deficient neutrophils also harbored reduced intracellular levels of G6P, lactate, and ATP.
Consistent with these findings, immunofluorescence analysis
using an antibody to GLUT1 showed that the expression of
membrane associated GLUT1 was markedly decreased in human
G6PC3-deficient neutrophils, compared with that in the control
subjects (Figure 5C).
We then examined the expression of gp91phox and p47phox
subunits of NADPH oxidase in neutrophils of G6PC3-deficient
patients and control subjects. Immunofluorescence analysis using
an antibody to gp91phox (Figure 6A) or p47phox (Figure 6B) showed
that the expression of the membrane associated NADPH oxidase
subunits was markedly decreased in neutrophils of both P1 and P2,
compared with the HD.
Discussion
Neutrophils require a constant supply of glucose for function and
survival, but have increased demand for glucose when responding
to immune threats requiring increased mobility, phagocytosis, and
Figure 6. Analysis of the expression of NADPH oxidase in
neutrophils of human G6PC3-deficient patients. Annexin V–
depleted blood neutrophils were isolated from HD (n ⫽ 5; ages 23
to 37 years) and 2 G6PC3-deficient patients, P1 and P2 as
described in “Depletion of apoptotic cells from neutrophils.” Two
separate experiments were conducted, each analyzed in duplicates. (A) Representative immunofluorescence analysis of
gp91phox (red fluorescence) and DAPI nuclei staining (blue fluorescence) at magnifications of 630⫻. (B) Representative immunofluorescence analysis of p47phox (green fluorescence), pan Cadherin
membrane staining (red fluorescence), and DAPI nuclei staining
(blue fluorescence) at 630⫻ magnification.
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2790
JUN et al
BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
Figure 7. Proposed pathways for G6P metabolism in normal
and G6PC3-deficient neutrophils. Glucose transported into the
cytoplasm via GLUT1 is metabolized by hexokinase to G6P
which can participate in glycolysis, hexose monophosphate shunt
(HMS), glycogen synthesis, or be translocated into the lumen of
the ER by the G6PT. In normal neutrophils, G6P localized within
the ER lumen can be hydrolyzed by G6Pase-␤ and the resulting
glucose transported back into the cytoplasm to reenter any of the
previously mentioned cytoplasmic pathways. However, in G6PC3deficient neutrophils, which lack a functional G6Pase-␤, ERlocalized G6P cannot be recycled to the cytoplasm. The GLUT1
transporter, responsible for the transport of glucose in and out of
the cell, is shown embedded in the plasma membrane. The
G6PT, responsible for the transport of G6P into the ER and
G6Pase-␤, responsible for hydrolyzing G6P to glucose and
phosphate, are shown embedded in the ER membrane.
ROS production. The current view is that there are 2 primary
pathways that compete for intracellular glucose/G6P in neutrophils: glycolysis and HMS.11 Within this view, the finding that there
are significant neutrophil defects in the absence of an ER compartmentalized G6Pase-␤ enzyme is not readily explained. Therefore
we hypothesized that ER uptake of G6P, to generate endoluminal
glucose by G6Pase-␤, must be a third major pathway, previously
overlooked, that competes for the utilization of cytoplasmic
glucose/G6P in neutrophils. We envisage that in a normal neutrophil, cytoplasmic G6P is transported into the ER, where it
accumulates until it is hydrolyzed by G6Pase-␤ to glucose, which
is then released back into the cytoplasm, either to be recycled to the
ER or converted by hexokinase into G6P for the glycolytic, HMS,
and/or glycogen synthetic pathways (Figure 7). Accordingly, in
G6PC3-deficient neutrophils, disruption of this recycling would
lead to accumulation of G6P in the ER, but prevent release of
glucose back to the cytoplasm (Figure 7). The resulting limitation
of cytoplasmic glucose/G6P availability would impact the cytoplasmic pathways of glucose metabolism and in turn impair additional
blood glucose uptake. To examine this hypothesis we examined
neutrophils extracted from G6pc3⫺/⫺ mice and their control
littermates and correlated those results to findings from neutrophils
from 2 human G6PC3-deficient patients. Both human and mouse
G6PC3-deficient neutrophils show similar results, suggesting they
are representative of the human disorder.
In the absence of G6Pase-␤, neutrophils were unable to acquire
sufficient exogenous glucose to meet cytoplasmic demand. Glucose uptake measurements confirmed this and correlated the lower
glucose uptake, reduced GLUT1 expression, and an impairment of
translocation of GLUT1 to the plasma membrane. As expected, the
lower glucose uptake resulted in a lower intracellular G6P concentration in both human and murine G6PC3-deficient neutrophils.
With less cytoplasmic glucose/G6P available, glycolysis would be
expected to be reduced, and this was also demonstrated by the
lower levels of lactate and ATP in human and murine G6PC3deficient neutrophils, which in turn further impair GLUT1 translocation and glucose uptake. Similarly the lower level of available
G6P would be expected to impact the HMS, leading to a reduction
in NADPH oxidase activity, as was also observed in human and
murine G6PC3-deficient neutrophils.
It is not clear to what relative extent the glycolytic, HMS, and
our proposed ER pathway compete for available cytoplasmic
glucose/G6P. Indeed, the relative importance of these pathways
might depend upon the needs of the cell at any given time. Existing
data suggest that 85% of the exogenous glucose taken up by the
neutrophils is metabolized via glycolysis to lactate.11,18 In this
study, we showed that the levels of lactate in murine and human
G6PC3-deficient neutrophils were 37% and 40% to 57%, respec-
tively, of those in control neutrophils, which might suggest that the
ER pathway competes for similar amounts of G6P as glycolysis.
This is strengthened by the observation that the levels of G6P,
lactate, and ATP in G6pc3⫺/⫺ neutrophils, which were 35%, 37%,
and 69%, respectively, of the levels in the control neutrophils, are
all coordinately reduced. Supporting this, the levels of G6P, lactate,
and ATP in human G6PC3-deficient neutrophils were 38% to 43%,
40% to 57%, and 68% to 76%, respectively, of the levels in
neutrophils of control subjects, are also coordinately reduced.
The G6PC3-deficient patients reported in this study were
identified based on clinical presentation of neutropenia and increased visibility of superficial veins, followed by sequence
confirmation that the G6PC3 gene is mutated. There have been
conflicting reports about the bone marrow in G6PC3-deficient
patients. The human and murine G6PC3-deficient bone marrows
reported in this study contain abundant mature neutrophils, which
contrasts to the G6PC3-deficient patients reported by Boztug et al14
showing an absence of mature neutrophils in the bone marrow.
Whether this reflects a true variability in the genotype-phenotype
relationship in the newly described disorder of G6PC3 deficiency is
very difficult to access until more patients are characterized. The
differences could reasonably be a reflection of the patients’ ages.
Bone marrows of the patients of Boztug et al14 were 1 to 2 months
old when characterized while G6PC3-deficient patients reported
here were significantly more mature—9 and 13 years of age. There
are many differences that could emerge during maturation of the
immune system that may contribute to the apparent heterogeneity
of the myeloid phenotype of G6PC3-deficient patients. On the
other hand, phenotypic heterogeneity in humans could account for
such disparities. It is also important to bear in mind that the
G6PC3-deficient patients we studied were being treated by G-CSF
therapy, while the G6pc3⫺/⫺ mice did not receive G-CSF treatment.
To delineate the mechanism underlying cellular function of G6PC3deficient neutrophils, it would be ideal to compare neutrophil
populations that have not been exposed to G-CSF, however this is
not ethically acceptable for human patients given the current
clinical standard of care. Therefore, it remains a possibility that
G-CSF treatment might also impact cellular functions in a severe
congenital neutropenia syndrome like G6PC3 deficiency. Given
these precautions, it is important to note that the present study
focused on the functional analysis of neutrophils, rather than the
nature of the bone marrow composition. In this respect, the
functional characteristics of human and mouse G6PC3-deficient
neutrophils are similar. We show that in nonapoptotic human and
mouse G6PC3-deficient neutrophils, G6Pase-␤ is essential for
normal energy homeostasis and a G6Pase-␤ deficiency prevents
recycling of ER glucose to the cytoplasm, leading to neutrophil
dysfunction.
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BLOOD, 14 OCTOBER 2010 䡠 VOLUME 116, NUMBER 15
NEUTROPHIL DYSFUNCTION IN G6PC3 DEFICIENCY
The phagocyte NADPH oxidase and ROS production play a key
role in host defense against microbial pathogens. The oxidase,
which is in a resting state in circulating blood neutrophils, can be
activated by a large variety of soluble and particulate agents.21,22
This activation process depends upon the phosphorylation and
membrane translocation of p47phox.42 G6P is absolutely required for
neutrophil respiratory burst activity, serving as the substrate of
G6PDH for NADPH production.11,19 Moreover, activation of
NADPH oxidase by agonists requires energy generated by glycolysis.43,44 We showed that the expression of gp91phox and p47phox is
markedly reduced in human and murine G6PC3-deficient neutrophils, compared with the controls. While PMA induces membrane
translocation of p47phox in control neutrophils, this agonist fails to
activate p47phox in G6pc3⫺/⫺ neutrophils. In conclusion, the decrease in
the expression and activation of NADPH oxidase results in
impairment of the respiratory burst activity in G6pc3⫺/⫺ neutrophils.
The expression of NADPH oxidase subunits can be stimulated
by glutamine45,46 and peroxisome proliferators-activated receptoralpha (PPAR-␣).47 In neutrophils, glutamine stimulates the expression of NADPH oxidase subunits gp91phox, p22phox, and p47phox and
is important for ROS production.45 Glutamine is a highly abundant
amino acid in the blood, largely synthesized in the skeletal muscle
by glutamine synthetase from glutamate, ammonia, and ATP.46,48
The G6Pase-␤/G6PT complex is also expressed in high levels in
the skeletal muscle, and generates endogenous glucose,49 a precursor of ATP. In G6PC3 deficiency, inactivation of G6Pase-␤ could
hamper muscle glucose production, leading to reduced glutamine
in both the muscle and the blood. This in turn would decrease
glutamine levels in neutrophils, resulting in reduced expression of
gp91phox, p22phox, and p47phox. Our preliminary results showed that
blood glutamine levels in wild type (n ⫽ 6) and G6pc3⫺/⫺ (n ⫽ 6)
mice were 1.25mM (⫾ 0.12mM) and 0.88mM (⫾ 0.05mM),
respectively, suggesting that inactivation of G6Pase-␤ reduced
glutamine in the blood. PPAR-␣ is a nuclear receptor belonging to a
superfamily of ligand-activated transcription factors that control
lipid, glucose and energy metabolism.50 In macrophages, PPAR-␣
agonists induce the expression of gp91phox and p47phox mRNA
expression and membrane p47phox protein.47 In pancreatic ␤ cells,
high glucose reduces PPAR-␣ mRNA expression.51 In future
studies we will investigate if reduced intracellular G6P/glucose in
neutrophils can decrease PPAR-␣ expression, resulting in downregulation of the expression of NADPH oxidase subunits, and the
role of glutamine in regulation of the expression of NADPH
oxidase in G6PC3 deficiency.
ATP plays a critical role in chemotaxis. Neutrophils release ATP
from the leading edge of the cell surface to amplify chemotactic
signals and direct cell orientation by feedback through P2 purinergic receptors.52 The released ATP can be hydrolyzed by ecto-
2791
ATPases to generate adenosine that regulates chemotaxis of
neutrophils by activating purinergic P1 receptors that are recruited
to the leading edge, to promote cell migration.53 ATP can increase
intracellular calcium levels in neutrophils, potentiating the oxidative burst and enhancing neutrophil adhesion to the endothelium.54
Moreover, ATP-mediated calcium sequestration by the ER or
sarcoplasmic reticulum is enhanced by G6P.55 Taken together the
ER-associated glucose/G6P reservoir plays a critical for energy
homeostasis and normal function of neutrophils.
The coupled action of G6Pase-␤ and G6PT to regulate the
cytoplasmic pathways of glycolysis and HMS suggests that GSD-Ib
neutrophils should exhibit similar defects. Indeed, the rate of 2-DG
uptake into human GSD Ib neutrophils is impaired30 and their
intracellular G6P levels are markedly lower, compared with the
control neutrophils,56 indicating that G6PT-deficient neutrophils
also manifest disrupted energy homeostasis.
In conclusion, we have shown that ER-localized G6Pase-␤
expression is important for exogenous glucose uptake across the
plasma membrane in annexin V–depleted nonapoptotic neutrophils
and is linked to their demand for cytoplasmic glucose/G6P.
G6Pase-␤ inactivation disrupts neutrophil energy homeostasis,
leading to impaired neutrophil respiratory burst, chemotaxis,
calcium flux, and phagocytosis. Our findings support the hypothesis that G6Pase-␤–dependent glucose/G6P recycling occurs in the
neutrophil and is a critical regulatory pathway for intracellular
glucose homeostasis.
Acknowledgments
This research was supported by the Intramural Research Programs
of the National Institute of Child Health and Human Development
and the National Institute of Allergy and Infectious Diseases,
National Institutes of Health.
Authorship
Contribution: H.S.J. designed and performed research, analyzed
data, and contributed to writing the paper; Y.M.L., Y.Y.C., D.H.M.,
and S.S.D. performed research; P.M.M. contributed to writing the
paper; B.C.M. analyzed data and contributed to writing the paper;
and J.Y.C. designed the research, analyzed data, and wrote the
paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Janice Y. Chou, Bldg 10, Rm 9D42, NIH,
10 Center Dr, Bethesda, MD 20892-1830; e-mail: chouja@mail.
nih.gov.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2010 116: 2783-2792
doi:10.1182/blood-2009-12-258491 originally published
online May 24, 2010
Lack of glucose recycling between endoplasmic reticulum and
cytoplasm underlies cellular dysfunction in glucose-6-phosphatase- β−
deficient neutrophils in a congenital neutropenia syndrome
Hyun Sik Jun, Young Mok Lee, Yuk Yin Cheung, David H. McDermott, Philip M. Murphy, Suk See De
Ravin, Brian C. Mansfield and Janice Y. Chou
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