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Molecular Genetics of Heritable Human Disorders

Janice Y. Chou, PhD, Head, Section on Cellular Differentiation
Hyun-Sik Jun, PhD, Postdoctoral Fellow
Young Mol Lee, PhD, Visiting Fellow
Brian C. Mansfield, PhD, Guest Researcher
Paul A. Mead, BS, Postbaccalaureate Fellow
Chi-Jiunn Pann, BS, Senior Research Assistant
Wentao Peng, PhD, Staff Scientist

Glycogen storage disease type I (GSD-I) comprises a group of autosomal recessive disorders consisting of GSD-Ia, which is caused by a deficiency in the liver-/kidney-/intestine-restricted glucose-6-phosphatase-alpha (G6Pase-alpha), and GSD-Ib, which is caused by a deficiency in a ubiquitously expressed glucose-6-phosphate transporter (G6PT). Normally, G6Pase-alpha hydrolyzes G6P to glucose, and G6PT translocates G6P from the cytoplasm to the lumen of the endoplasmic reticulum (ER). Both diseases manifest a phenotype of disturbed glucose homeostasis, although GSD-Ib patients also suffer from neutropenia and neutrophil dysfunctions. There is no cure for GSD-I, and the current dietary therapy cannot prevent the development of long-term complications. The recent development of animal disease models presents an opportunity to delineate the disease more precisely and develop therapies that target the underlying disease process. Recently, we reported a second ubiquitously expressed G6P hydrolase, G6Pase-beta (G6PC3). Neutrophils express both G6Pase-beta and G6PT, suggesting that neutrophil homeostasis and function may require endogenous glucose production in the ER via the concerted action of G6Pase-beta and G6PT. To examine this hypothesis, we generated mouse lines deficient in either G6Pase-beta or G6PT and showed that both G6Pase-beta–deficient (G6pc3⁻^/⁻) and G6PT-deficient mice manifest neutropenia and neutrophil dysfunctions that mimic the myeloid symptoms of human GSD-Ib patients.

Neutrophil stress and apoptosis underlie myeloid dysfunction in GSD-Ib

GSD-Ib patients and mice manifest neutropenia and neutrophil dysfunctions of unknown mechanism. Neutrophils express both G6PT and G6Pase-beta that together transport G6P into the ER lumen and hydrolyze it to glucose. Given that G6PT-deficient neutrophils should be unable to produce endogenous glucose, we hypothesized that such an inability would lead to ER stress and increased apoptosis. Using GSD-Ib mice, we showed that GSD-Ib neutrophils exhibit elevated production of ER chaperones and oxidative stress, consistent with ER stress, and increased Annexin V binding and caspase-3 activation, consistent with an increased rate of apoptosis. Bax activation, mitochondrial release of pro-apoptotic effectors, and caspase-9 activation demonstrate the involvement of the intrinsic mitochondrial pathway in these processes. The results show that G6P translocation and hydrolysis are required for normal neutrophil function, supporting the hypothesis that neutrophil dysfunction in GSD-Ib is attributable, at least in part, to ER stress and increased apoptosis.

The angiotensin system mediates renal fibrosis in GSD-Ia nephropathy

Despite intensive dietary therapy, GSD-Ia patients develop renal disease of unknown etiology. We showed that the expression of angiotensinogen (Agt), angiotensin (Ang) type 1 receptor, transforming growth factor-β1 (TGF-beta1), and connective tissue growth factor (CTGF) was higher in the kidneys of GSD-Ia mice than in controls. Renal expression of Agt rose one week earlier than that of TGF-beta1 and CTGF, consistent with the up-regulation of TGF-beta1 and CTGF by Ang II. Renal fibrosis was characterized by a marked increase in the synthesis and deposition of extracellular matrix proteins in the renal cortex and histological abnormalities that included tubular basement membrane thickening, atrophy, dilation, and multifocal interstitial fibrosis. We further elucidated the mechanism of renal disease by showing that the Ang/TGF-beta1 pathway also elicits renal damage through oxidative stress. Our results indicate that activation of the Ang/TGF-beta1 pathway plays an important role in the pathophysiology of renal disease in GSD-Ia.

Hepatic injury correlates with increased neutrophil infiltration of the liver in GSD-Ia

GSD-Ia patients manifest hepatocellular adenoma (HCA) of unknown etiology. We showed that both GSD-Ia patients and mice have an underlying immune abnormality characterized by subclinical neutrophilia and elevated serum chemokines IL-8 or KC. We further showed that the elevation in serum IL-8 was more prominent in HCA-bearing GSD-Ia patients. Correlated with this elevation, in the mouse model of GSD-Ia, we observed hepatic injury characterized by necrotic foci and increased chemokines KC and MIP-2 as well as elevated neutrophil infiltration of the liver, suggesting one mechanism by which adenoma may arise.

The G6PT is a phosphate-linked antiporter that is deficient in GSD-Ib and GSD-Ic

It is well established that a deficiency in G6PT causes GSD-Ib. Interestingly, deleterious mutations in the G6PT gene were identified in clinical cases of GSD-Ic proposed to be deficient in an inorganic phosphate (P_i) transporter. We hypothesized that G6PT is both the G6P and P_i transporter. Using reconstituted proteoliposomes, we showed that both G6P and P_i are efficiently taken up into P_i-loaded G6PT-proteoliposomes but uptake is not detectable in P_i-loaded proteoliposomes containing the p.R28H G6PT null mutant. The G6PT-proteoliposome–mediated G6P or P_i uptake is inhibited by cholorgenic acid and vanadate, both specific G6PT inhibitors. Taken together, our results suggest that G6PT has a dual role as a G6P and a P_itransporter and that GSD-Ib and GSD-Ic are deficient in the same G6PT gene.

Both G6PT and the bacterial hexose-6-phosphate transporter UhpT are phosphate- (P_i) linked antiporters. We previously characterized G6PT mutations by measuring G6P uptake activities in microsomes co-expressing G6PT and G6Pase-alpha. We have developed a new assay, based on reconstituted proteoliposomes carrying only G6PT, and characterized G6P and P_i uptake activities of 23 G6PT mutations. We showed that co-expression and G6PT-only assays are equivalent in measuring G6PT activity. We further addressed a concern about the structure of G6PT. Protease protection and glycosylation scanning assays have suggested that G6PT is anchored to the ER by 10 transmembrane domains. However, recent homology modeling proposed that G6PT may contain 12 helices and that amino acids essential for the functions of UhpT also play important roles in G6PT. Site-directed mutagenesis and in vitro expression assays demonstrated that only one of the four residues critical for UhpT activity is essential in G6PT. Furthermore, glycosylation scanning and protease sensitivity assays showed that the 10-domain model of G6PT is more probable than the 12-domain UhpT-like model.

Neutrophils deficient in G6Pase-beta undergo accelerated apoptosis via the mitochondrial stress pathway

Neutrophils differentiate and mature in the bone marrow (BM). We have previously shown that G6pc3^−/−mice lacking G6Pase-bata (G6PC3) manifest neutropenia and that activated G6pc3^−/− neutrophils, isolated from peritoneal exudates, exhibit dysfunction, increased production of ER chaperones, and enhanced apoptosis. Using resting neutrophils from the BM, we showed that G6pc3^−/−BM is neutropenic and that the resting neutrophils exhibit impaired respiratory burst, chemotactic, and calcium flux activities. Moreover, G6pc3^−/−BM neutrophils exhibit ER stress, oxidative stress, and ultrastructural alterations of the ER. Activation of the protein kinase–like ER kinase (PERK) along with increased expression of phosphorylated eukaryotic translation initiation factor 2α, activating transcription factor 4, and C/EBP-homologous protein demonstrate the involvement of the PERK–mediated ER stress signaling pathway. G6pc3^−/−BM neutrophils exhibit increased Annexin V binding and caspase-3 activation, consistent with an increased rate of apoptosis. Bax activation, mitochondrial release of pro-apoptotic effectors, and caspase-9 activation demonstrate the involvement of the intrinsic mitochondrial apoptotic pathway. Taken together, the results demonstrate a critical role for G6Pase-beta in ER homeostasis and normal neutrophil functions.

Normoglycemia alone is insufficient to prevent hepatocellular adenoma, a long-term complication of GSD-Ib

GSD-Ib patients develop the 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 by 2 weeks after 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 and 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 to be at risk for this long-term complication.

Generation of mice with a conditional allele for G6pc

For improve our understanding of the roles of G6Pase-alpha (G6PC) in different tissues and in pathological conditions, we 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 in the examination of the consequence of tissue-specific G6Pase-alpha deficiency and the mechanisms of long-term complications in GSD-Ia.

Publications

Kim SY, Jun HS, Mead PA, Mansfield BC, Chou JY. Neutrophil stress and apoptosis underlie myeloid dysfunction in glycogen storage disease type Ib. Blood 2008 111:5704-5711.
Yiu WH, Pan C-J, Ruef RA, Peng WT, Starost MF, Mansfield BC, Chou JY. The angiotensin system mediates renal fibrosis in glycogen storage disease type Ia nephropathy. Kidney Int 2008 73:716-723.
Chen SY, Pan CJ, Nandigama K, Mansfield BC, Ambudkar SV, Chou JY. The glucose-6-phosphate transporter is a phosphate-linked antiporter deficient in glycogen storage disease type Ib and Ic. FASEB J 2008 22:2206-2213.
Yiu WH, Pan CJ, Mead PA, Starost MF, Mansfield BC, Chou JY. Normoglycemia alone is insufficient to prevent long term complications of hepatocellular adenoma in glycogen storage disease type Ib mice. J Hepatol 2009 51:909-917.
Peng WT, Pan CJ, Lee EJ, Westphal H, Chou JY. Generation of mice with a conditional allele for G6pc. Genesis 2009 47:590-594.

Collaborators

Suresh V. Smbudkar, PhD, Laboratory of Cell Biology, Center for Cancer Research, NCI, Bethesda, MD
Eric J. Lee, DVM, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Krishnamachary Nandigama, PhD, Laboratory of Cell Biology, Center for Cancer Research, NCI, Bethesda, MD
Matthew F. Starost, PhD, DVR, Division of Veterinary Resources, NIH, Bethesda, MD
David A. Weinstein, MD, University of Florida College of Medicine, Gainesville, FL
Heiner Westphal, MD, Program in Genomics of Differentiation, NICHD, Bethesda, MD

Contact

For further information, contact chouja@mail.nih.gov.

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