Glycogen storage disease type I (GSD-I) is caused by deficiencies in the glucose-6-phosphatase-alpha (G6Pase-alpha) complex expressed primarily in the liver, kidney, and intestine. The complex is consisted of a glucose-6-phosphate transporter (G6PT1) that translocates glucose-6-phosphate (G6P) from cytoplasm to the lumen of the endoplasmic reticulum (ER) and G6Pase-alpha that hydrolyses G6P to glucose. Together, they maintain interprandial glucose homeostasis. Deficiencies in G6Pase-alpha causes GSD-Ia and deficiencies in G6PT cause GSD-Ib. Both manifest the symptoms of G6Pase-alpha deficiency characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. GSD-Ib patients also present with neutropenia and myeloid dysfunctions. The current dietary therapy can not treat the underlying disease and long-term complications develop in adult patients. While the molecular basis for the GSD-I disorders are now known, many aspects of the diseases are still poorly understood. Recent development of animal disease models now opens the opportunity to delineate the disease more precisely and to develop therapies targeting the underlying disease process. Until recently, G6Pase activity was considered confined to the liver, kidney and intestine, the only tissues known to contain the G6Pase-alpha catalytic unit. However, there were some inconsistencies in this view, the most notably being that several studies suggested that GSD-Ia patients are still capable of producing endogenous glucose even when the G6Pase-alpha complex is disrupted. This led to our discovery of a second G6P hydrolase, G6Pase-beta that couples with G6PT1 to form an active G6Pase complex in the same way as G6Pase-alpha. Our findings challenge the current dogma that only liver, kidney and intestine can contribute to blood glucose homeostasis. G6Pase-alpha is a highly hydrophobic protein anchored to the ER by 9-transmembrane helices. The protein can not be expressed in a soluble form, therefore, enzyme replacement therapy is not an option for the treatment of GSD-Ia. To be functional, G6Pase-alpha must not only embed correctly in the ER membrane, but also couple with G6PT1. Therefore, somatic gene therapy, targeting a G6Pase-alpha gene to the liver and the kidney, is an attractive possibility for treating GSD-Ia. We have employed G6Pase-alpha-/- mice that manifest symptoms characteristic of humans GSD-1a to develop gene replacement therapies for GSD-Ia. Previously, we showed that infusion of an adeno-associated virus (AAV) serotype 2 vector carrying murine G6Pase-alpha (AAV2-G6Pase-alpha) into neonatal GSD-Ia mice failed to sustain their life beyond weaning. To improve the efficacy of rAAV-mediated gene transfer for GSD-Ia, we evaluated two new AAV serotypes, AAV serotype 1 (AAV1) and AAV serotype 8 (AAV8). Both are reported to direct more efficient hepatic gene transfer than the AAV2. We showed that neonatal infusion of G6Pase-alpha-/- mice with the AAV1-G6Pase-alpha or AAV8-G6Pase-alpha resulted in hepatic expression of the G6Pase-alpha transgene and markedly improved the survival of the mice. However, only the AAV1-G6Pase-alpha could achieve significant renal transgene expression. We therefore devised a more effective strategy, in which a neonatal AAV1-G6Pase-alpha infusion was followed by a second infusion at age one week. This approach provided sustained expression of a complete, functional, G6Pase-alpha system in both the liver and kidney and corrected the murine GSD-Ia disorder for the full 57 weeks of the study. This type of approach, which is effective in correcting the metabolic imbalances and disease progression in GSD-Ia mice hold promise for the future of gene therapy in humans. Glucose is absolutely essential for the survival and function of the brain. In our current understanding, there is no endogenous glucose production in the brain and it is totally dependent upon blood glucose. This glucose is generated, between meals, by the hydrolysis of G6P in the liver and the kidney. The demonstration of a significant, specific, G6P hydrolase that can couple to G6PT1 outside of the liver raises interesting questions about the ability of other tissues to cycle glucose and contribute to blood glucose homeostasis. We show that astrocytes, the main reservoir of brain glycogen, express both G6Pase-beta and G6PT1 and that they can couple to form an active G6Pase complex. One role of astrocytes is to buffer the brain during periods of prolonged sleep deprivation, seizures, or mild hypoxia. The presence of an active G6Pase complex in astrocytes suggests that these cells can also provide an endogenous source of brain glucose. The islet-specific G6Pase-related protein (IGRP) was first characterized as an islet-specific phosphohydrolase based on its sequence similarity to G6Pase-alpha, but IGRP is devoid of phosphohydrolase activity. Recently, amino acids 206 to 214 in IGRP were identified as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes. This suggests that amino acids 206 to 214 in IGRP confer functional specificity to IGRP. We therefore investigated the molecular events that inactivate IGRP activity and the effects of the beta cell antigen sequence on the stability and enzymatic activity of G6Pase-alpha. We showed that the residues responsible for G6Pase-alpha activity are more extensive than previously recognized. Introducing the IGRP antigenic motif into G6Pase-alpha did not completely destroy activity, although it did destabilize the protein. Instead we showed that the low hydrolytic activity in IGRP is due to the combination of multiple independent mutations. We also showed that G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, which prevents the targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells dictate susceptibilities to diabetes. GSD-Ia patients manifest a pro-atherogenic lipid profile characterized by hypercholesterolemia, hypertriglyceridemia, reduced cholesterol in HDL, and increased cholesterol in LDL and VLDL fractions but are not at elevated risk for developing atherosclerosis. One explanation for this may be attributable to reverse cholesterol transport. This process, which recycles cholesterol from peripheral tissues to the liver, is protective against atherosclerosis and is stimulated by HDL. Using G6Pase-alpha-/- mice, which exhibit a typical GSD-Ia pro-atherogenic lipid profile, we examined this possibility by measuring the efficacy of sera from G6Pase-alpha-/- mice in promoting cellular efflux of free cholesterol, the first step in reverse cholesterol transport. Serum phospholipid, which correlates positively with the scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux, and apolipoprotein A-IV and E, acceptors for ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol transport, are increased in GSD-Ia mice. We show that both SR-BI- and ABCA1-mediated effluxes are more efficient in the presence of sera from G6Pase-alpha-/- mice than sera of control littermates. Since the potential of serum to promote cellular cholesterol efflux is inversely correlated with atherosclerosis, these observations provide one explanation why GSD-Ia patients are apparently protected against premature atherosclerosis.
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