Treating neutropenia and neutrophil dysfunction in glycogen storage disease type Ib with an SGLT2 inhibitor

Abstract

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)-deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.

Graphical abstract

Figure 1.

Empagliflozin lowers 1,5AG plasma levels and restores neutrophil function in GSD-Ib patients by lowering neutrophil 1,5AG6P. 1,5AG is a nondegradable glucose analog present in blood (∼150 µM). It is slowly phosphorylated to 1,5AG6P by the side activities of hexokinases and adenosine 5′-diphosphate–dependent glucokinase present in neutrophils. To prevent its accumulation, 1,5AG6P is transported into the endoplasmic reticulum by G6PT and dephosphorylated by the phosphatase G6PC3. (A) In GSD-Ib patients who are deficient in G6PT, 1,5AG6P accumulates in neutrophils. It is the rise in the concentration of 1,5AG6P that intoxicates neutrophils by strongly inhibiting hexokinases and depleting the intracellular pool of G6P (Gluc-6P) that is vital for neutrophils to survive and function. (B) Inhibiting the renal SGLT2 with empagliflozin leads to glucosuria by preventing the renal reabsorption of glucose, but also of 1,5AG, which results in its urinary excretion. Consequently, this leads to an approximate fourfold reduction in the concentration of 1,5AG in blood and of 1,5AG6P in neutrophils. This relieves the inhibition of hexokinases and increases the pool of G6P and of the metabolites in downstream pathways, improving glycolysis, respiratory burst, and protein glycosylation. Neutrophils function better, and neutropenia is partly corrected.

Figure 2.

Empagliflozin lowers plasma 1,5AG and intracellular 1,5AG6P and corrects neutropenia in GSD-Ib patients. (A) Time course of ANCs before and during empagliflozin treatment in 4 GSD-Ib patients. The top part of the right scale refers to the concentration of GCSF, and the bottom to empagliflozin. (B) Decrease in the concentration of plasma 1,5AG determined by liquid chromatography–mass spectrometry (LC-MS) during treatment. (C) Neutrophil 1,5AG6P determined in blood cells after centrifugation to remove plasma (for determination of 1,5AG) and normalized to total metabolite content (total ion current [TIC]) and ANC. (D) 1,5AG6P in isolated granulocytes (polymorphonuclear neutrophils [PMNs]) and peripheral blood mononuclear cells (PBMCs) obtained from patient 1 (PT1) 1 day before and 28 or 50 days into empagliflozin treatment and compared with levels found in a healthy control (CT).

Figure 3.

Empagliflozin corrects protein glycosylation and oxidative burst in GSD-Ib patients after empagliflozin treatment. (A) Role for G6P (Gluc-6P) in uridine diphosphate glucose (UDP-Gluc) production (essential for protein glycosylation) and reduced NAD phosphate (NADPH) production (essential for the respiratory burst reactions). (B) Western blots illustrating the almost complete correction of glycosylation for the protein LAMP2 in granulocytes (PMNs) from PT1 and PT3 isolated before and during empagliflozin treatment and compared with a healthy control (CT). (C) Empagliflozin treatment corrected the defective respiratory burst in PT4. Whole blood was stimulated with phorbol myristate acetate (PMA) at 37°C for 15 minutes. Red blood cells were lysed, and the sample was analyzed immediately by flow cytometry with gating set on neutrophils. Gate B: neutrophils showing increased DHR123 fluorescence compared with background fluorescence in unstimulated cells. Gate C: PT4 PMA-stimulated neutrophils showing DHR123 fluorescence equivalent to that of the date-matched normal control. ADP, adenosine 5′-diphosphate; ATP, adenosine triphosphate; MW, molecular weight; PPi, inorganic pyrophosphate; UTP, G1P uridylyltransferase.

Figure 3.

Empagliflozin corrects protein glycosylation and oxidative burst in GSD-Ib patients after empagliflozin treatment. (A) Role for G6P (Gluc-6P) in uridine diphosphate glucose (UDP-Gluc) production (essential for protein glycosylation) and reduced NAD phosphate (NADPH) production (essential for the respiratory burst reactions). (B) Western blots illustrating the almost complete correction of glycosylation for the protein LAMP2 in granulocytes (PMNs) from PT1 and PT3 isolated before and during empagliflozin treatment and compared with a healthy control (CT). (C) Empagliflozin treatment corrected the defective respiratory burst in PT4. Whole blood was stimulated with phorbol myristate acetate (PMA) at 37°C for 15 minutes. Red blood cells were lysed, and the sample was analyzed immediately by flow cytometry with gating set on neutrophils. Gate B: neutrophils showing increased DHR123 fluorescence compared with background fluorescence in unstimulated cells. Gate C: PT4 PMA-stimulated neutrophils showing DHR123 fluorescence equivalent to that of the date-matched normal control. ADP, adenosine 5′-diphosphate; ATP, adenosine triphosphate; MW, molecular weight; PPi, inorganic pyrophosphate; UTP, G1P uridylyltransferase.