Glycogen storage disease type I (GSD-I) is caused by deficiencies in the glucose-6-phosphatase-alpha (G6Pase-alpha) complex, which consists of glucose-6-phosphate transporter (G6PT1) and G6Pase-alpha. In the nondisease state, G6PT1 translocates G6P from cytoplasm to the lumen of the endoplasmic reticulum, and G6Pase-alpha hydrolyzes G6P to glucose; together, G6PT1 and G6Pase-alpha maintain glucose homeostasis. On the other hand, deficiencies in G6Pase-alpha and G6PT1 cause GSD-Ia and GSD-Ib, respectively. Patients with both GSD-Ia and GSD-Ib manifest the symptoms of G6Pase-alpha deficiency, which is characterized by hypoglycemia and hepatomegaly. GSD-Ib patients also present with myeloid dysfunctions. The current dietary therapy cannot treat the underlying disease, and long-term complications develop in adult patients. Recently developed animal models provide an opportunity to delineate the disease more precisely and to develop therapies. Until recently, G6Pase activity was believed to be confined to the liver, kidney, and intestine, the only tissues known to contain G6Pase-alpha. However, several studies suggest that GSD-Ia patients are still capable of producing endogenous glucose even when the G6Pase-alpha complex is disrupted. These studies led to our discovery of a second G6P hydrolase, G6Pase-beta, that couples with G6PT1 to form an active G6Pase complex, as does G6Pase-alpha. Our findings challenge the current dogma that only liver, kidney, and intestine can contribute to blood glucose homeostasis.
Recombinant adeno-associated virus-mediated gene therapy for murine GSD-Ia
Ghosh, Allamarvdasht, Pan, Sun,1 Mansfield, Chou; in collaboration with Byrne
G6Pase-alpha is a highly hydrophobic protein anchored to the endoplasmic reticulum (ER) by nine-transmembrane helices. The protein cannot 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 it must 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. To that end, we employed G6Pase-alpha–/– mice, which manifest symptoms characteristic of human 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 the mice 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 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 the kidney and corrected the murine GSD-Ia disorder for the full 57 weeks of the study (Sun et al., Hum Mol Genet 2002;11:2155). Such an approach, which is effective in correcting the metabolic imbalances and disease progression in GSD-Ia mice, holds promise for the future of gene therapy in humans.
Ghosh A, Allamarvdasht M, Pan C-J, Sun M-S, Mansfield BC, Byrne BJ, Chou JY. Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer. Gene Ther 2005 [Epub ahead of print].
Active brain G6Pase-beta complex capable of endogenous glucose production
Ghosh, Cheung, Mansfield, Chou
Glucose is absolutely essential for the survival and function of the brain. According to our current understanding, no endogenous glucose production occurs in the brain, and the organ is totally dependent on blood glucose. Between meals, glucose is generated 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 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 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 also provide an endogenous source of brain glucose.
Ghosh A, CheungYY, Mansfield BC, Chou JY. Brain contains a functional glucose-6-phosphatase complex capable of endogenous glucose production. J Biol Chem 2005;280:11114-11119.
Shieh J-J, Pan C-J, Mansfield BC, Chou JY. Glucose-6-phosphate hydrolase, widely expressed outside the liver, can explain age-dependent resolution of hypoglycemia in glycogen storage disease type Ia. J Biol Chem 2003;278:47098-47103.
Islet-specific G6Pase-related protein
2 Pan, Mansfield, Chou
Based on its sequence similarity to G6Pase-alpha, the islet-specific G6Pase-related protein (IGRP) was first characterized as an islet-specific phosphohydrolase; however, IGRP is devoid of phosphohydrolase activity. Recently, others identified amino acids 206 to 214 in IGRP as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes, suggesting 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. We showed that the low hydrolytic activity in IGRP is instead attributable to a combination of several independent mutations. We also showed that G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, preventing the targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells dictate susceptibilities to diabetes.
Shieh J-J, Pan C-J, Mansfield BC, Chou JY. In islet-specific glucose-6-phosphatase-related protein, the beta cell antigenic sequence that is targeted in diabetes is not responsible for the loss of phosphohydrolase activity. Diabetologia 2005;48:1851-1859.
Shieh J-J, Pan C-J, Mansfield BC, Chou JY. The islet-specific glucose-6-phosphatase-related protein, implicated in diabetes, is a glycoprotein embedded in the endoplasmic reticulum membrane. FEBS Lett 2004;562:160-164.
Increased cellular cholesterol efflux in murine GSD-Ia
Nguyen, Pan, Shieh,2 Chou
While 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, they are not at elevated risk for developing atherosclerosis. One explanation may be reverse cholesterol transport, a process that recycles cholesterol from peripheral tissues to the liver, protects against atherosclerosis, and is stimulated by HDL. Using G6Pase-alpha–/– mice, which exhibit a typical GSD-Ia pro-atherogenic lipid profile, we examined such a 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. In GSD-Ia mice, we observed an increase in serum phospholipid, which correlates positively with the scavenger receptor class B type I (SR-BI)–mediated cholesterol efflux, as well as an increase in apolipoprotein A-IV and E, acceptors for ATP-binding cassette transporter A1 (ABCA1)–mediated cholesterol transport. We showed 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. Given that 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.
Nguyen AD, Pan C-J, Shieh J-J, Chou JY. Increased cellular cholesterol efflux in glycogen storage disease type Ia mice: a potential mechanism that protects against premature atherosclerosis. FEBS Lett 2005;579:4713-4723.
1Mao-Sen Sun, MD, former Postdoctoral Fellow
2Jeng-Jer Shieh, PhD, former Postdoctoral Fellow
COLLABORATOR
Barry J. Byrne, MD, University of Florida, Gainesville, FL
For further information, contact chouja@mail.nih.gov.