Mesenchymal Stem Cells Lacking Glucose-6-Phosphatase-β Exhibit Disturbed Energy Homeostasis and Defective Adipogenesis

Glucose-6-phosphatase-β (G6Pase-β or G6PC3) is a ubiquitously expressed glucose-6-phosphate (G6P) hydrolase that catalyzes the hydrolysis of G6P to glucose and phosphate. It is a key enzyme for intracellular glucose production in non-gluconeogenic organs. Mutations in G6Pase-β underlie severe congenital neutropenia syndrome type 4 (SCN4, MIM612541), an autosomal recessive disease characterized by neutropenia and neutrophil dysfunction. A mouse model for G6Pase-β deficiency ( G6pc3 -/- ) established by gene targeting manifests both neutropenia and neutrophil dysfunction, mimicking the human disorder. Recent studies have shown that in G6Pase-β deficiency enhanced neutrophil apoptosis leads to neutropenia while disrupted energy homeostasis underlies neutrophil dysfunction. The patients of G6Pase-β deficiency also present with non-haematological defects, including prominent superficial venous pattern, congenital cardiac anomaly, myopathy, disrupted bone remodeling, and genital anomalies, suggesting that G6Pase-β deficiency leads to a broader cell dysfunction. We now hypothesize that these non-haematological defects may reflect loss of glucose metabolism within mesenchymal stem cells (MSC) that require levels of glucose beyond those supplied by the blood. MSC are multipotent stem cells that are capable of differentiating into all connective tissue types including bone, cartilage, myocytes, and adipocytes. Studies have shown that MSC cultured in reduced glucose concentrations exhibit increased proliferative ability and reduced replicative senescence. Using MSC isolated from the compact bones, we show that G6pc3 -/- MSC grow at a significantly faster rate than those of wild type MSC under both normoxia and hypoxia conditions. Consistently, the colony-forming unit-fibroblastic counts in G6pc3 -/- MSC are 2-fold higher than those in wild type MSC, suggesting that G6pc3 -/- MSC contain higher numbers of progenitors. Differentiation of MSC involves metabolic shifts between glycolysis and mitochondrial oxidative phosphorylation. An increase in the ratio of lactate to pyruvate correlates with increased glycolysis and an increase in oxygen consumption measures increased mitochondrial oxidative phosphorylation. Adipogenesis and osteogenesis of MSC have been shown to be associated with increased oxygen consumption, while chondrogenesis of MSC is associated with increased glycolysis. We now show that cellular levels of lactate and ATP in G6pc3 -/- MSC are 4.5- and 2-fold higher, respectively than those in wide type MSC. Moreover, G6pc3 -/- MSC exhibit a 2.5-fold increase in the ratio of lactate to pyruvate but a decrease in oxygen consumption, compared to wild type MSC, suggesting that G6Pase-β-deficient MSC exhibit enhanced glycolysis along with impaired mitochondrial respiration activity. This also suggests that adipogenesis and osteogenesis of MSC may be impaired in G6pc3 -/- MSC. Adipogenic differentiation of MSC from mice was then evaluated by morphological alterations and lipid accumulation. MSC from wild type mice, cultured for 3 days in adipogenic differentiation medium, changed from fibroblast-like cells to round-shaped adipocyte-like cells, while G6pc3 -/- MSC cultured under the same conditions did not show any morphological changes. Wild type MSC, cultured for 5 days in adipogenic differentiation medium, yielded many adipocytes containing lipid droplets and staining positive by Oil red O, while adipocytes were rarely seen in G6pc3 -/- MSC cultured under similar conditions, confirming the impaired adipogenic differentiation of G6pc3 -/- MSC. In conclusion, the non-hematological defects associated with G6Pase-β deficiency may result from altered energy homeostasis in G6pc3 -/- MSC, leading to enhanced glycolysis along with impaired mitochondrial respiration activity, that impacts MSC differentiation. Disclosures No relevant conflicts of interest to declare.