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. ? ? Previous views held that there was a single ER enzyme, G6Pase-alpha, whose activity, limited to the liver, kidney and intestine, was solely responsible for hydrolysis of G6P to glucose for release to the blood. Recently, we characterized a second ubiquitously expressed G6Pase activity, G6Pase-beta (G6PC3) that shares kinetic properties, topological and active site structure to G6Pase-alpha and couples functionally with the G6PT to form an active G6Pase complex that hydrolyses G6P to glucose. This suggests that the G6Pase-beta-G6PT complex might be functional in neutrophils and that the myeloid defects in GSD-Ib are due to the loss of activity of that complex. To test this, we generated G6Pase-beta-deficient (G6pc3-/-) mice and show that G6pc3-/- mice manifest neutropenia and neutrophil dysfunctions mimicking GSD-Ib. We further show that neutrophils from G6pc3-/- mice undergo ER stress and enhanced rate of apoptosis. The results demonstrate that G6P translocation and metabolism in the ER are critical for normal neutrophil functions and define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology.? ? GSD-Ia patients develop renal disease of unknown etiology despite intensive dietary therapies. We show that the expression of angiotensinogen (Agt), angiotensin type 1 receptor, transforming growth factor-beta1 (TGF-beta1), 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-beta1 and CTGF, consistent with the up-regulation of TGF-beta1 and CTGF by angiotensin 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. Our results suggest that activation of the angiotensin system plays an important role in the pathophysiology of renal disease in GSD-Ia. ? ? GSD-Ia patients manifest hepatocellular adenoma (HCA) of unknown etiology. We show that both GSD-Ia patients and mice have an underlying immune abnormality characterized by a sub-clinical neutrophilia and elevated serum chemokine IL-8 or KC. We further show that the elevation in serum IL-8 is more prominent in HCA-bearing GSD-Ia patients. Correlated with this, in the mouse model of GSD-Ia we observe hepatic injury characterized by necrotic foci, increased hepatic chemokines, KC and MIP-2, and increased neutrophil infiltration of the liver, suggesting one mechanism by which adenoma may arise. ? ? ? 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.? ? GSD-Ib patients and mice manifest neutrppenia 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. 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.
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