Failure to eliminate a phosphorylated glucose analog leads to neutropenia in patients with G6PT and G6PC3 deficiency

Abstract

Neutropenia represents an important problem in patients with genetic deficiency in either the glucose-6-phosphate transporter of the endoplasmic reticulum (G6PT/SLC37A4) or G6PC3, an endoplasmic reticulum phosphatase homologous to glucose-6-phosphatase. While affected granulocytes show reduced glucose utilization, the underlying mechanism is unknown and causal therapies are lacking. Using a combination of enzymological, cell-culture, and in vivo approaches, we demonstrate that G6PT and G6PC3 collaborate to destroy 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a close structural analog of glucose-6-phosphate and an inhibitor of low-K_M hexokinases, which catalyze the first step in glycolysis in most tissues. We show that 1,5AG6P is made by phosphorylation of 1,5-anhydroglucitol, a compound normally present in human plasma, by side activities of ADP-glucokinase and low-K_M hexokinases. Granulocytes from patients deficient in G6PC3 or G6PT accumulate 1,5AG6P to concentrations (∼3 mM) that strongly inhibit hexokinase activity. In a model of G6PC3-deficient mouse neutrophils, physiological concentrations of 1,5-anhydroglucitol caused massive accumulation of 1,5AG6P, a decrease in glucose utilization, and cell death. Treating G6PC3-deficient mice with an inhibitor of the kidney glucose transporter SGLT2 to lower their blood level of 1,5-anhydroglucitol restored a normal neutrophil count, while administration of 1,5-anhydroglucitol had the opposite effect. In conclusion, we show that the neutropenia in patients with G6PC3 or G6PT mutations is a metabolite-repair deficiency, caused by a failure to eliminate the nonclassical metabolite 1,5AG6P.

Keywords: 1,5-anhydroglucitol; SLGT2 inhibitors; glucose-6-phosphatase-β; metabolite repair; neutropenia.

Fig. 1.

Substrate specificity of G6PC1, G6PC3, and the glucose-6-phosphate transporter. (A) Wild-type (WT) and catalytically inactive (H176A or H167A) human G6PC1 and G6PC3 were produced as recombinant proteins with a C-terminal 6×His tag in HEK293T cells. Equal amounts of the membrane fraction were analyzed by Western blotting with anti-6×His antibody. (B) The membrane preparations were used at appropriate concentrations to assay the phosphatase activity on the indicated substrates (all tested at 100 µM) through the release of inorganic phosphate. Phosphatase activity observed in preparations from cells expressing the catalytically dead mutants was assessed under identical conditions and subtracted to account for baseline phosphatase activity. (C) Ribose-5-P and glucose-6-P phosphatase activities were assayed with a radiochemical assay (10 µM) in rat microsomes from the indicated tissues. (D) Ratios of the ribose-5-P and glucose-6-P phosphatase activities measured in C are compared with those measured with membrane preparations from HEK293T cells overexpressing recombinant G6PC3 or G6PC1. (E) Hydrolysis of the best substrates (100 µM) for G6PC3 was assayed in rat skeletal muscle microsomes in the presence or absence of the G6PT inhibitor S3483 (100 µM) and of 7.5 mM octyl glucoside (OG). For B and C, data are means and error bars are ±SEM (n = 3). For E, data are means and error bars are ±SD (n ≥ 4). P values (ns, not significant, P > 0.05; *P ≤ 0.05; ^$P ≤ 0.0001) were determined with unpaired t tests using the two-stage linear step-up procedure of Benjamini et al. (51), with Q = 1%.

Fig. 2.

1,5-Anhydroglucitol-6-phosphate accumulates in human HAP1 cell lines deficient in G6PC3 or G6PT grown in the presence of 1,5-anhydroglucitol and leads to inhibition of glycolysis and of the pentose phosphate pathway, and cell death. Wild-type, G6PT-deficient (clone A4), and G6PC3-deficient (clones D7 and A6) HAP1 cells were cultured for 24 h in DMEM (1 g/L glucose) with or without addition of the indicated concentrations of 1,5AG (A and B) or 1,5-anhydrofructose (C–H). Growth medium was replaced after 20 h and ∼4 h before the preparation of cell extracts. The indicated intracellular metabolites were analyzed by LC-MS (A, C, and E–H). Glucose consumption was calculated from the remaining glucose in the medium after 20 h (B and D). LC-MS measurements were normalized to the untreated conditions (=100%) within the experiment (E–H). Values represent means ± SD of three independent experiments. (I) Cell survival was measured after 72-h growth in the same media (means ± SD; n = 3). Statistical analysis: (B and D) P values were determined using multiple unpaired t tests (for each cell line the treated condition was compared with the untreated), and statistical significance was determined using the Holm–Sidak method, with alpha = 0.05. (A, C, and E–I) P values were determined with two-way ANOVA, and statistical significance was determined using the post hoc Dunnett’s test for multiple comparisons [comparison of KO with wild type with the same treatment (A, C, and E–H); comparison of the treated with the untreated condition (I)], with alpha = 0.05. *P ≤ 0.05; ^∋P ≤ 0.01; ^∞P ≤ 0.001; ^$P ≤ 0.0001. 1,5-AF, 1,5-anhydrofructose; TIC, total ion current.

Fig. 3.

Toxicity of physiological concentrations of 1,5-anhydroglucitol in an immortalized mouse neutrophil precursor cell line deficient in G6PC3. (A) Cell survival of estrogen-dependent immortalized mouse G6PC3-deficient neutrophil progenitors (ER-Hoxb8) was measured after 72-h culture in the presence of the indicated concentrations of 1,5AG or 1,5-anhydrofructose (means ± SD; n = 3). (B) Glycolytic glucose utilization was indirectly evaluated by measuring the detritiation of 2-[³H]glucose (at the phosphoglucose isomerase step) in the presence or absence of 0.2 mM 1,5AG (means ± SD; n = 3). (C–H) Intracellular metabolites were analyzed by LC-MS/MS in extracts prepared from wild-type and G6PC3-deficient cells at different time points after addition of 0.2 mM 1,5AG (means ± SD; n = 6). Statistical analysis: (A) P values were determined with two-way ANOVA, and statistical significance was determined using the Dunnett’s post hoc test for multiple comparisons (comparison of the treated with the untreated condition), with alpha = 0.05. (B–H) P values were determined with multiple unpaired t tests (B: For each time point, one data point in one condition was compared with data points from the three other conditions; C–H: For each time point, the G6PC3 KO was compared with the WT), and statistical significance was determined using the Holm–Sidak method, with alpha = 0.05. *P ≤ 0.05; ^∋P ≤ 0.01; ^∞P ≤ 0.001; ^$P ≤ 0.0001. DHAP, dihydroxyacetone-phosphate; F1,6P₂, fructose-1,6-bisphosphate; G6P, glucose-6-phosphate; GA3P, glyceraldehyde-3-phosphate.

Fig. 4.

Modulating the concentration of 1,5-anhydroglucitol in blood impacts neutrophils in G6PC3-deficient mice. G6PC3 KO or heterozygous littermates (8 to 11 wk old; 20 to 24 g) were left untreated or treated with 100 µL of an empagliflozin (EMPA) suspension (10 mg⋅kg⁻¹; 10 times over 14 d) or 1,5AG (100 µL of a 50 mM solution; five times from day 7) until euthanasia on day 14 (n = 6 to 8 for each group). (A and B) 1,5AG in serum was measured at the indicated time points by LC-MS analysis (A) or from plasma recovered from EDTA-blood collected after euthanasia (B). (C) Flow cytometry analysis with anti–Mac-1 (CD11b-PE) and anti–Gr-1 (Ly-6G-FITC) antibodies was performed to quantify granulocytes. (D) LC-MS analysis of 1,5AG6P in extracts from isolated white blood cells (WBCs). (E–H) Bone marrow smears from 1,5AG-treated G6PC3 heterozygous (E and G) or knockout (F and H) mice were stained with May–Grünwald (E and F) or peroxidase (G and H), showing a maturation arrest with accumulation of apparent promyelocytes (“P”) in G6PC3 KO mice (F and H) and normal maturation with plenty of mature neutrophils (“N”) in heterozygous mice (E and G), and confirming the myeloid nature of these cells (peroxidase-positive in G and H). (Scale bar, 20 μm.) Statistical analysis: (A–D) P values were determined with one-way ANOVA followed by multiple t tests using the Bonferroni correction, with alpha = 0.05. Data are means ± SD (n = 6 to 8). ns, not significant, P > 0.05; ^∋P ≤ 0.01; ^∞P ≤ 0.001; ^$P ≤ 0.0001. (Magnification: G, 100×.)

Fig. 5.

1,5-Anhydroglucitol-6-phosphate accumulates in neutrophils from patients deficient in G6PT or G6PC3. (A) Concentration of 1,5AG6P was determined by LC-MS in granulocytes (PMNs) and lymphocytes (PBMCs) obtained from patients and healthy controls. (B) Serum 1,5AG in two GSDIb (filled symbols) and one G6PC3-deficient (open symbols) patients and seven healthy controls (CT) was determined by LC-MS (each symbol represents a different control; for some individuals, blood samples were taken on two different occasions to estimate variability and the two values are shown). PT2: GSDIb is treated with G-CSF.

Fig. 6.

Illustration of the role played by 1,5-anhydroglucitol-6-phosphate accumulation in the neutropenia found in G6PC3 and G6PT deficiency. 1,5AG (also called 1-deoxyglucose) is a polyol that resembles glucose and is normally present in blood. It is transported into neutrophils and slowly phosphorylated by a side activity of low-K_M hexokinases (HK1, HK2, and HK3) and ADP-dependent glucokinase to 1,5AG6P. The glucose-6-P transporter of the endoplasmic reticulum (G6PT) transports 1,5AG6P into the ER, where it is dephosphorylated by G6PC3. When patients are deficient in G6PT or G6PC3, 1,5AG6P accumulates in neutrophils to concentrations that strongly inhibit low-K_M hexokinases, the enzymes catalyzing the first step of glycolysis. This depletes the intracellular pool of glucose-6-P in granulocytes, which likely decreases ATP production in glycolysis, as well as likely NADPH production in the pentose-P pathway and also UDP-glucose availability for protein glycosylation, explaining neutrophil dysfunction and neutropenia described in these patients.