Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice

Abstract

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.

Keywords: Genetic diseases; Glucose metabolism; Hematology; Mouse models.

Figure 1. Generation of a G6PD-deficient mouse model.

(A) Schematic representation of the WT G6PD locus (top) and related modifications (bottom). (B) Predicted protein sequence of knocked-in G6PD gene. (C) G6PD activity in RBCs from WT versus G6PD_Med- mice. n = 5–7 mice per group. Unpaired, 2-tailed t test. (D) Mouse versus human mRNA levels in G6PD_Med- and WT mice; assay 1 detects both WT and G6PD_Med- mRNA, assay 2 detects only WT, and assays 3 and 4 detect only G6PD_Med- mRNA (details of assay primers and probes shown in Supplemental Figure 2A). n = 5 for both groups for the Pol2RA experiment; n = 3 for WT and G6PD. Data shown as mean ± SD in C and D. (E) Western blot analysis of cytoplasm from RBCs. Because the G6PD gene is X linked, males were used in all experiments. G6PD_Med- mice are hemizygous for the knocked-in human gene; WT mice are littermate controls with the mouse WT G6PD. n = 3 for each group.

Figure 2. Metabolic effect of diamide challenge in RBCs from WT and G6PD_Med- mice.

RBCs from WT and G6PD_Med- mice were incubated with 1 mM diamide (A). RBCs were tested at 0 minutes (no diamide), 30 minutes, 1 hour, 2 hours, 6 hours, and 12 hours from incubation with diamide prior to metabolomics analysis. Multivariate analyses including PLS-DA (B) and hierarchical clustering analysis (C) clearly indicate a time-dependent effect of the treatment on RBCs (PC1 explaining 45.5% of the total variance) and highlight the impact of G6PD activity (PC3 explaining 10.7% of the total variance). Significant metabolites by repeated measures 1-way ANOVA are shown in the heatmap in C. (D) The experiment was repeated by incubating RBCs in the presence of [1,2,3-¹³C₃]-glucose. By quantifying isotopologs M+2 and M+3 of lactate (and the relative ratio), fluxes through PPP versus glycolysis can be determined (E — median ± range) and confirm a significantly lower activation of this pathway in RBCs from G6PD_Med- mice upon diamide challenge. n = 4 for both groups. **P < 0.01, ***P < 0.001. EMP, Embden-Meyerhof-Parnas.

Figure 3. Overview of glycolysis, PPP, glutathione metabolism and recycling, and purine oxidation in RBCs from WT (shown in blue) and G6PD_Med- (shown in red) mouse RBCs (n = 3) treated with 1 mM diamide for up to 12 hours.

Time points tested were 0 minutes (no diamide), 30 minutes, 1 hour, 2 hours, 6 hours, and 12 hours from incubation with diamide. Line plots indicate median ± range per each time point. Y axes indicate metabolite abundance in arbitrary units. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Phenylhydrazine induces brisk hemoglobinuria in G6PD-deficient mouse RBCs but not in WT.

(A) RBC life span in mice at baseline (without any oxidative stress). (B) Biotinylation experiments showed significant decreases in circulating RBC survival after the second PHZ treatment in G6PD_Med- mice. (C) Hemoglobinuria was observed in G6PD_Med-. but not WT mice after PHZ treatment. (D) G6PD_Med- RBCs at baseline and upon treatment with PHZ for up to 7 days are incapable of activating the PPP, as determined by the ratios of G6PD product/substrate, a proxy for the determination of the enzyme activity by law of mass action, as described (16) (E — median ± range). Y axis indicates glucose-6-phosphate (G6P) to 6-phospho-gluconate (6PG) ratios. n = 4 per group. (F) Pathway analysis highlighted 4 PPP-related metabolites on the top 5 significant metabolic changes by 2-way ANOVA, as a function of G6PD status (blue dots) or PHZ treatment (red dots). (G) Significant metabolic differences 24 hours after PHZ treatment.

Figure 5. Metabolic impact of PHZ treatment in vivo on WT and G6PD-deficient mouse RBCs.

Overview of glycolysis, PPP, glutathione metabolism and recycling, and purine oxidation in RBCs from WT (blue) and G6PD_Med- (red) mice (n = 3) treated with 1 mM PHZ for up to 7 days. Time points tested were 0 (no PHZ), 1, 2, 5, and 7 days from treatment with PHZ. Line plots indicate median ± range per each time point. Y axes indicate metabolite abundance in arbitrary units. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. G6PD-deficient mouse RBCs have comparable metabolism and posttransfusion recovery to WT RBCs at the end of storage.

RBCs from WT and G6PD_Med- mice (n = 6) were stored under conditions mimicking storage in the blood bank for 12 days (A). At the end of storage, RBCs were transfused into UbiC-GFP C57BL/6-Tg(UBC-GFP)30Scha/J (Ubi-GFP) mice, and flow cytometry studies were performed to determine the percentage of transfused RBCs circulating at 24 hours from transfusion, which was determined to be comparable between the 2 groups. Transfusing G6PD_Med- GFP into WT or G6PD_Med- non-GFP recipients did not impact the posttransfusion recovery (PTR) percentage measurement. (B) Metabolic phenotypes of G6PD_Med- RBCs showed some significant differences at baseline (especially with respect to glycolysis, the PPP, and glutathione homeostasis). However, these changes were not appreciable by the end of storage. (C) Individual data points for G6P and 6PG are shown (significance calculated by Mann-Whitney unpaired t test). *P < 0.05, **P < 0.01.

Figure 7. Metabolic analyses of brain, heart, and kidney from WT and G6PD_Med- mice.

(A) Significant metabolic changes were observed in all the organs tested from transgenic mice, compared with WT controls, n = 4 for both groups. (B–D) as highlighted by multivariate principal component and hierarchical clustering analyses (only the top 25 significant metabolites by t test are shown).

Figure 8. Metabolic analyses of liver and spleen from WT and G6PD_Med- mice.

(A) Significant metabolic changes were observed in all the organs tested from G6PD_Med- mice, compared with WT controls, (B and C) as highlighted by multivariate principal component and hierarchical clustering analyses (only the top 25 significant metabolites by t test are shown). The majority of metabolic changes in organs of G6PD_Med- mice are related to fatty acid metabolism and acyl-carnitines, followed by amino acid metabolism and glycolysis. Lactate levels were significantly lower in all organs of G6PD_Med- mice. n = 4 for both groups. (D) Individual data points are shown for lactate in each organ in WT (blue bars) and G6PD_Med- (red bars). (E) Organ-to-organ metabolic differences overweighed differences between WT and G6PD_Med- mice.