The Section on Cellular differentiation conducts research to understand the biology and pathophysiology of GSD-I and G6Pase-beta deficiency and to develop novel therapeutic approaches. GSD-Ia patients develop renal disease of unknown etiology despite intensive dietary therapies. We show that the expression of angiotensinogen (Agt), angiotensin (Ang) type 1 receptor, transforming growth factor-1 (TGF-1), and connective tissue growth factor (CTGF) were elevated in the kidneys of GSD-Ia mice compared to the controls. The increase in renal expression of Agt was one week earlier than the increase in TGF-1 and CTGF, consistent with the up-regulation of TGF- 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 including tubular basement membrane thickening, atrophy, dilation, and multifocal interstitial fibrosis. We further elucidate the mechanism of renal disease by showing that the Ang/TGF-1 pathway also elicits renal damage through oxidative stress. Our results indicate that activation of the Ang/TGF-1 pathway plays an important role in the pathophysiology of renal disease in GSD-Ia. 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. It has been 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 (Pi) transporter. We hypothesized that G6PT is both the G6P and Pi transporter. Using reconstituted proteoliposomes we show that both G6P and Pi are efficiently taken up into Pi-loaded G6PT-proteoliposomes but is not detectable in Pi-loaded proteoliposomes containing the p.R28H G6PT null mutant. The G6PT-proteoliposomes-mediated G6P or Pi 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 Pi transporter 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 (Pi)-linked antiporters. We previously characterized G6PT mutations by measuring G6P uptake activities in microsomes co-expressing G6PT and G6Pase-alpha. We now report a new assay, based on reconstituted proteoliposomes carrying only G6PT, and characterize G6P and Pi uptake activities of 23 G6PT mutations. We show that co-expression and G6PT-only assays are equivalent in measuring G6PT activity. We further address 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. 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. GSD-Ib patients and mice manifest neutropenia and neutrophil dysfunctions of unknown mechanism. Neutrophils express both G6PT and G6Pase- that together transport G6P into the ER lumen and hydrolyze it to glucose. Since G6PT-deficient neutrophils are expected to be unable to produce endogenous glucose, we hypothesized this would lead to ER stress and increased apoptosis. Using GSD-Ib mice, we show that GSD-Ib neutrophils exhibit increases in the production of ER chaperons 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 demonstrate that G6P translocation and hydrolysis are required for normal neutrophil functions and support the hypothesis that neutrophil dysfunction in GSD-Ib is due, at least in part, to ER stress and increased apoptosis. 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 now show 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 2, 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.
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