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. GSD-Ib patients develop a long-term complication of hepatocellular adenomas (HCA). To evaluate whether maintaining normoglycemia in GSD-Ib could prevent HCA, we infused neonatal GSD-Ib mice with adeno-associated virus (AAV) carrying G6PT and examined their metabolic and myeloid phenotypes for the 72-week study. The AAV vector delivered the G6PT transgene to the liver and bone marrow. Long term metabolic correction was achieved alongside a transient myeloid correction. Hepatic G6PT activity was 50% of wild-type levels at 2 weeks post-infusion but declined rapidly thereafter to reach 3% of wild-type levels by age 6 to 72 weeks. Despite this, the infused mice maintained normoglycemia throughout the study, exhibited near normal growth and normalized serum metabolite profiles. However, all five AAV-treated GSD-Ib mice that lived over 50 weeks accumulated excessive hepatic glycogen and fat. Two mice developed steatohepatitis and multiple HCAs with one undergoing malignant transformation. The results suggest normoglycemia alone cannot prevent hepatic steatosis and glycogen accumulation or the development of HCAs in GSD-Ib, providing one explanation why GSD-Ib patients maintaining normoglycemia under intense dietary therapy continue at risk for this long-term complication. 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. For better understanding of the roles of G6Pase-alpha (G6PC) in different tissues and in pathological conditions, we have generated mice harboring a conditional null allele for G6pc by flanking exon 3 of the G6pc gene with loxP sites. We confirmed the null phenotype by using the EIIa-Cre transgenic approach to generate mice lacking exon 3 of the G6pc gene. The resulting homozygous Cre-recombined null mice manifest a phenotype mimicking G6Pase-alpha-deficient mice and human GSD-Ia patients. This G6pc conditional null allele will be valuable to examine the consequence of tissue-specific G6Pase-alpha deficiency and the mechanisms of long-term complications in GSD-Ia. 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. GSD-Ia patients manifest impaired glucose homeostasis with long-term renal disease. We have previously shown that renal fibrosis in GSD-Ia is mediated by the angiotensin/transforming growth factor-beta1 (TGF-beta1) pathway which also elicits renal damage through oxidative stress. We now elucidate the mechanism of renal disease by showing that renal expression of Nox-2, p22phox, and p47phox, components of NADPH oxidase, are up-regulated in GSD-Ia mice compared to controls. Akt/protein kinase B, a downstream mediator of angiotensin II and TGF-beta1, is also activated, leading to phosphorylation and inactivation of the Forkhead box O family of transcription factors. This in turn triggers down-regulation of superoxide dismutase and catalase activities that play essential roles in oxidative detoxification in mammals. Renal oxidative stress in GSD-Ia mice is demonstrated by increased oxidation of dihydroethidium and by oxidative damage of DNA. Importantly, renal dysfunction, reflected by elevated serum levels of blood urea nitrogen, reduced renal catalase activity, and increased renal fibrosis, is improved in GSD-Ia mice treated with the antioxidant drug tempol. These data provide the first evidence that oxidative stress is one mechanism that underlies GSD-Ia nephropathy.
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