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, 116 (15), 2783-92

Lack of Glucose Recycling Between Endoplasmic Reticulum and Cytoplasm Underlies Cellular Dysfunction in glucose-6-phosphatase-beta-deficient Neutrophils in a Congenital Neutropenia Syndrome

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Lack of Glucose Recycling Between Endoplasmic Reticulum and Cytoplasm Underlies Cellular Dysfunction in glucose-6-phosphatase-beta-deficient Neutrophils in a Congenital Neutropenia Syndrome

Hyun Sik Jun et al. Blood.

Abstract

G6PC3 deficiency, characterized by neutropenia and neutrophil dysfunction, is caused by deficiencies in the endoplasmic reticulum (ER) enzyme glucose-6-phosphatase-β (G6Pase-β or G6PC3) that converts glucose-6-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-β in neutrophils remains unknown. We hypothesized that the ER recycles G6Pase-β-generated glucose to the cytoplasm, thus regulating the amount of available cytoplasmic glucose/G6P in neutrophils. Accordingly, a G6Pase-β deficiency would impair glycolysis and hexose monophosphate shunt activities leading to reductions in lactate production, adenosine-5'-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 G6pc3(-/-) mice and G6PC3-deficient patients along with impaired glucose uptake in G6pc3(-/-) 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 p47(phox) are down-regulated in murine and human G6PC3-deficient neutrophils. The results establish that in nonapoptotic neutrophils, G6Pase-β is essential for normal energy homeostasis. A G6Pase-β deficiency prevents recycling of ER glucose to the cytoplasm, leading to neutrophil dysfunction.

Figures

Figure 1
Figure 1
G6pc3−/− 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 β-actin. Data from 2 pairs of littermates are shown and each lane contains 80 μg of protein. (C) A plot of BM neutrophil counts in control and G6pc3−/− mice determined by flow cytometry analysis using anti–Gr-1 and anti-CD11b antibodies (n = 4). (D) Western blot analysis of protein extracts of neutrophils using antibodies against GRP78, GRP170, PDI, or β-actin. Data from 2 pairs of littermates are shown and each lane contains 50 μg 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.
Figure 2
Figure 2
Annexin V–depleted BM neutrophils from G6pc3−/− mice exhibit impaired function. The 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) 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.
Figure 3
Figure 3
Analysis of 2-DG uptake, the expression of GLUT1, and intracellular G6P, lactate, ATP, and glycogen levels in G6pc3−/− 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 RPMI-1640 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 β-actin. Each lane contains 50 μg 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.
Figure 4
Figure 4
Analysis of the expression of NADPH oxidase in G6pc3−/− 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 β-actin. Data from 2 pairs of littermates are shown and each lane contains 50 μg 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.
Figure 5
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 glucose-containing 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.
Figure 6
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.
Figure 7
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-β and the resulting glucose transported back into the cytoplasm to reenter any of the previously mentioned cytoplasmic pathways. However, in G6PC3-deficient neutrophils, which lack a functional G6Pase-β, ER-localized 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-β, responsible for hydrolyzing G6P to glucose and phosphate, are shown embedded in the ER membrane.

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