The Section on Cellular differentiation conducts research to understand the biology and pathogenesis of GSD-I and G6Pase-beta deficiency and to develop novel therapeutic approaches for these disorders. G6PC3 deficiency, characterized by neutropenia and neutrophil dysfunction, is caused by deficiencies in G6Pase-beta (or G6PC3) that converts 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-beta in neutrophils remains unknown. We hypothesized that the ER recycles G6Pase-beta-generated glucose to the cytoplasm, thus regulating the amount of available cytoplasmic glucose/G6P in neutrophils. Accordingly, a G6Pase-beta deficiency would impair glycolysis and hexose monophosphate shunt activities leading to reductions in lactate production, ATP production, and NADPH oxidase activity. Using non-apoptotic 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 to their respective controls. In parallel, the expression of NADPH oxidase subunits and membrane translocation of p47phox are down-regulated in murine and human G6PC3-deficient neutrophils. The results establish that in non-apoptotic neutrophils, G6Pase-beta is essential for normal energy homeostasis. A G6Pase-beta deficiency prevents recycling of ER glucose to the cytoplasm, leading to neutrophil dysfunction. G6PC3 or G6Pase-beta deficiency underlies a congenital neutropenia syndrome in which neutrophils exhibit enhanced ER stress, increased apoptosis, impaired energy homeostasis, and impaired functionality. We show that murine G6pc3-/- neutrophils undergoing ER stress activate protein kinase-like ER kinase and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3)/Akt signaling pathways, and that neutrophil apoptosis is mediated, in part, by the intrinsic mitochondrial pathway. In G6PC3-deficient patients, granulocyte colony stimulating factor (G-CSF) improves neutropenia but its impact on neutrophil apoptosis and dysfunction is unknown. We now show that G-CSF delays neutrophil apoptosis in vitro by modulating apoptotic mediators. However, G6pc3-/-neutrophils in culture exhibit accelerated apoptosis compared to wild type neutrophils both in the presence or absence of G-CSF. Limiting glucose (0.6 mM) accelerates apoptosis but is more pronounced for wild type neutrophils, leading to similar survival profiles for both neutrophil populations. In vivo G-CSF therapy completely corrects neutropenia and normalizes levels of p-Akt, PtdIns(3,4,5)P3, and active caspase-3. Neutrophils from in vivo G-CSF-treated G6pc3-/- mice exhibit increased glucose uptake and elevated intracellular levels of G6P, lactate, and ATP, leading to improved functionality. Together, the results strongly suggest that G-CSF improves G6pc3-/- neutrophil survival by modulating apoptotic mediators and rectifies function by enhancing energy homeostasis. GSD-Ia patients deficient in G6Pase-alpha manifest impaired glucose homeostasis. We examined the efficacy of liver G6Pase-alpha delivery mediated by AAV-GPE, an AAV serotype 8 vector expressing human G6Pase-alpha directed by the human G6PC promoter/enhancer (GPE), and compared it to AAV-CBA, that directed murine G6Pase-alpha expression using a hybrid chicken beta-actin (CBA) promoter/CMV enhancer. The AAV-GPE directed hepatic G6Pase-alpha expression in the infused G6pc-/- mice declined 12-fold from age 2 to 6 weeks but stabilized at wild type levels from age 6 to 24 weeks. In contrast, the expression directed by AAV-CBA declined 95-fold over 24 weeks, demonstrating that the GPE is more effective in directing persistent in vivo hepatic transgene expression. We further show that the rapid decline in transgene expression directed by AAV-CBA results from an inflammatory immune response elicited by the AAV-CBA vector. The AAV-GPE-treated G6pc-/- mice exhibit normal levels of blood glucose, blood metabolites, hepatic glycogen, and hepatic fat. Moreover the mice maintained normal blood glucose levels even after 6 hours of fasting. The complete normalization of hepatic G6Pase-alpha deficiency by the G6PC promoter/enhancer holds promise for the future of gene therapy in human GSD-Ia patients. Blood glucose homeostasis between meals depends upon production of glucose within the ER of the liver and kidney by hydrolysis of G6P into glucose and phosphate (Pi). This reaction depends on coupling the G6PT with G6Pase-alpha. Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a Pi-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:Pi exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-alpha. We show that SLC37A1 and SLC37A2 are ER-associated, Pi-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-alpha. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.
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