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. G6Pase-beta deficiency underlies a congenital neutropenia syndrome in which neutrophils exhibit enhanced apoptosis, impaired energy homeostasis, and impaired functionality. The ubiquitous pattern of G6Pase-beta expression suggests that G6Pase-βplays a critical role in non-gluconeogenic tissues where there may be increased demands for glucose. Macrophages play key roles in innate immunity, inflammation, and tissue remodeling. During pregnancy, macrophages also influence the homeostasis of the developing placenta, and are important in preventing premature fetal rejection. Macrophages have increased demand for glucose like neutrophils. Pregnancy is a condition with increased demand for glucose. We hypothesized that the cycling pathway for G6P metabolism also occurs in macrophages and pregnancy uterus and G6Pase-beta deficiency is associated with impaired energy homeostasis in both macrophages and pregnant uterus, resulting in macrophage dysfunction and pregnancy-associated complications. This was proven to be the case. We showed that murine G6pc3-/- macrophages exhibit impairments in their respiratory burst, chemotaxis, calcium flux, and phagocytic activities. Consistent with a G6P metabolism deficiency, G6pc3-/- macrophages also have a lower glucose uptake, and lower levels of G6P, lactate, and ATP than wild type macrophages. Furthermore, the expression and activation of NADPH oxidase activity is down-regulated. We further showed that during pregnancy, the absence of G6Pase-βactivity also impaired energy homeostasis in the uterus. Supporting this, G6pc3-/- macrophages exhibit repressed trafficking in vivo both during an inflammatory response and in pregnancy. The dysfunctional macrophages and impaired uterine energy homeostasis correlate with reduced fertility in G6pc3-/- mothers. GSD-Ia patients deficient in G6Pase-alpha manifest impaired glucose homeostasis. There is no cure for GSD-Ia, but many of the disease symptoms can be managed or improved using dietary therapies. While the therapies are sufficiently successful to enable patients to attain near normal growth and pubertal development, the underlying pathological process remains uncorrected. As a result, long-term complications including hepatocellular adenoma (HCA) with malignant potential still persist in GSD-Ia patients. Using our G6pc-/- mouse model of GSD-Ia, we examined the efficacy of liver G6Pase-alpha delivery mediated by rAAV8-GPE, a recombinant AAV serotype 8 vector expressing human G6Pase-alpha directed by the human G6PC promoter/enhancer (GPE) and showed that rAAV8-GPE-mediated gene transfer completely normalizes hepatic G6Pase-alpha deficiency for at least 24 weeks. A remaining concern with the therapy is the risk of HCA. A recent study showed that HCA develops in 100% of liver-specific G6pc-null mice 78 weeks after gene deletion. Therefore we examined the disease risk for hepatic neoplasia in a long-term study using G6pc-/- mice. We show that rAAV8-GPE-mediated gene therapy maintains efficacy for at least 70-90 weeks for mice expressing more than 3% of wild type hepatic G6Pase-alpha activity. The treated mice displayed normal hepatic fat storage, normal blood metabolite and glucose tolerance profiles, reduced fasting blood insulin levels, maintained normoglycemia over a 24-hour fast. Ultrasound and histological examinations have shown no hepatic steatosis nor detectable HCA or hepatocellular carcinoma in the liver of any of these mice. After a 24-hour fast, hepatic G6PT mRNA levels in rAAV8-GPE-treated G6pc-/- mice were markedly increased. Since G6PT transport is the rate-limiting step in microsomal G6P metabolism it may explain why the treated G6pc-/- mice could sustain prolonged fasts. The low fasting blood insulin levels and lack of hepatic steatosis may explain the absence of HCA. Our results suggest that G6Pase-alpha gene transfer may offer a therapeutic approach to the management of human GSD-Ia. To develop a gene therapy for GSD-Ia, rAAV vectors carrying G6Pase-αdirected by a variety of different promoter/enhancer elements have been investigated. The most efficacious vectors are rAAV8-GPE, a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer and rAAV8-miGPE, a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer. To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-αexpression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia. G6PT belongs to the SLC37 family of membrane-bound transporters. Only G6PT, also known as SLC37A4, has been characterized, and it acts as a Pi-linked G6P antiporter. The other three SLC37 family members 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. The activity of SLC37A3 is unknown. 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. GSD-Ib is characterized by impaired glucose homeostasis and immune deficiency. The underlying cause of GSD-Ib neutropenia is an enhanced neutrophil apoptosis but patients also manifest neutrophil dysfunction of unknown etiology. Previously, we showed G6PT interacts with the enzyme G6Pase-beta to regulate the availability of G6P/glucose in neutrophils. A deficiency in G6Pase-beta activity in neutrophils impairs both their energy homeostasis and function. We now show that G6PT-deficient neutrophils from GSD-Ib patients are similarly impaired. Their energy impairment is characterized by decreased glucose uptake and reduced levels of intracellular G6P, lactate, ATP, and NADPH, while functional impairment is reflected in reduced neutrophil respiratory burst, chemotaxis, and calcium mobilization. The transcription factor HIF-1αplays a vital role in the regulation of glycolytic capacity and energy metabolism in myeloid cells. HIF-1αis also an upstream activator of PPAR-γ, a nuclear receptor that regulates lipid and glucose metabolism as well as inflammation. We show that the HIF-1α/PPAR-γpathway is activated in G6PT-deficient neutrophils, leading to impairment in neutrophil respiratory burst, chemotaxis, and calcium mobilization activities. Together these findings show that the G6PT-mediated G6P/glucose cycling is essential for neutrophil homeostasis and G6P metabolism and a deficiency leads to impaired energy homeostasis. We further show that the mechanism of neutrophil dysfunction in GSD-Ib arises from activation of the HIF-1α/PPAR-γpathway.
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