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Brian C. Mansfield - One of the best experts on this subject based on the ideXlab platform.
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gene therapy using a novel g6pc s298c variant enhances the long term efficacy for treating glycogen storage disease type ia
Biochemical and Biophysical Research Communications, 2020Co-Authors: Lisa Zhang, Brian C. Mansfield, Javier Anduaga, Matthew F Starost, Cheol Woo Lee, Irina Arnaoutova, Janice Y. ChouAbstract:Abstract The current phase I/II clinical trial for human glycogen storage disease type-Ia (GSD-Ia) (NCT 03517085) uses a recombinant adeno-associated virus (rAAV) vector expressing a codon-optimized human glucose-6-phosphatase-α (G6Pase-α or G6PC). DNA sequence changes introduced by codon-optimization can negatively impact gene expression. We therefore generated a novel variant in which a single amino acid change, S298C, is introduced into the native human G6PC sequence. Short term gene transfer study in G6pc−/− mice showed that the rAAV-G6PC-S298C vector is 3-fold more efficacious than the native rAAV-G6PC vector. We have shown previously that restoring 3% of normal hepatic G6Pase-α activity in G6pc−/− mice prevents hepatocellular adenoma/carcinoma (HCA/HCC) development and that mice harboring
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glycogen storage disease type ia mice with less than 2 of normal hepatic glucose 6 phosphatase α activity restored are at risk of developing hepatic tumors
Molecular Genetics and Metabolism, 2017Co-Authors: Goo-young Kim, Brian C. Mansfield, Joonhyun Kwon, Jun-ho Cho, Young Mok Lee, Matthew F Starost, Chijiunn Pan, Janice Y. ChouAbstract:Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA) and carcinoma (HCC), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). We have previously shown that G6pc-/- mice receiving gene transfer mediated by rAAV-G6PC, a recombinant adeno-associated virus (rAAV) vector expressing G6Pase-α, and expressing 3-63% of normal hepatic G6Pase-α activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-α activity required to prevent tumor formation remained unknown. In this study, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-α and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-α expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3-33% of normal hepatic G6Pase-α activity (AAV mice) maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-α activity (AAV-low mice), 3 expressing 0.9-1.3% of normal hepatic G6Pase-α activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing ≥3% of normal hepatic G6Pase-α activity. The results establish the threshold of hepatic G6Pase-α activity required to prevent HCA/HCC and show that GSD-Ia mice harboring <2% of normal hepatic G6Pase-α activity are at risk of tumor development.
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type i glycogen storage diseases disorders of the glucose 6 phosphatase glucose 6 phosphate transporter complexes
Journal of Inherited Metabolic Disease, 2015Co-Authors: Janice Y. Chou, Brian C. Mansfield, Hyun Sik JunAbstract:Disorders of the glucose-6-phosphatase (G6Pase)/glucose-6-phosphate transporter (G6PT) complexes consist of three subtypes: glycogen storage disease type Ia (GSD-Ia), deficient in the liver/kidney/intestine-restricted G6Pase-α (or G6PC); GSD-Ib, deficient in a ubiquitously expressed G6PT (or SLC37A4); and G6Pase-β deficiency or severe congenital neutropenia syndrome type 4 (SCN4), deficient in the ubiquitously expressed G6Pase-β (or G6PC3). G6Pase-α and G6Pase-β are glucose-6-phosphate (G6P) hydrolases with active sites lying inside the endoplasmic reticulum (ER) lumen and as such are dependent upon the G6PT to translocate G6P from the cytoplasm into the lumen. The tissue expression profiles of the G6Pase enzymes dictate the disease's phenotype. A functional G6Pase-α/G6PT complex maintains interprandial glucose homeostasis, while a functional G6Pase-β/G6PT complex maintains neutrophil/macrophage energy homeostasis and functionality. G6Pase-β deficiency is not a glycogen storage disease but biochemically it is a GSD-I related syndrome (GSD-Irs). GSD-Ia and GSD-Ib patients manifest a common metabolic phenotype of impaired blood glucose homeostasis not shared by GSD-Irs. GSD-Ib and GSD-Irs patients manifest a common myeloid phenotype of neutropenia and neutrophil/macrophage dysfunction not shared by GSD-Ia. While a disruption of the activity of the G6Pase-α/G6PT complex readily explains why GSD-Ia and GSD-Ib patients exhibit impaired glucose homeostasis, the basis for neutropenia and myeloid dysfunction in GSD-Ib and GSD-Irs are only now starting to be understood. Animal models of all three disorders are now available and are being exploited to both delineate the disease more precisely and develop new treatment approaches, including gene therapy.
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functional analysis of mutations in a severe congenital neutropenia syndrome caused by glucose 6 phosphatase β deficiency
Molecular Genetics and Metabolism, 2015Co-Authors: Suru Lin, Brian C. Mansfield, Chijiunn Pan, Janice Yang ChouAbstract:Abstract Glucose-6-phosphatase-β (G6Pase-β or G6PC3) deficiency is characterized by neutropenia and dysfunction in both neutrophils and macrophages. G6Pase-β is an enzyme embedded in the endoplasmic reticulum membrane that catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. To date, 33 separate G6PC3 mutations have been identified in G6Pase-β-deficient patients but only the p.R253H and p.G260R missense mutations have been characterized functionally for pathogenicity. Here we functionally characterize 16 of the 19 known missense mutations using a sensitive assay, based on a recombinant adenoviral vector-mediated expression system, to demonstrate pathogenicity. Fourteen missense mutations completely abolish G6Pase-β enzymatic activity while the p.S139I and p.R189Q mutations retain 49% and 45%, respectively of wild type G6Pase-β activity. A database of residual enzymatic activity retained by the G6Pase-β mutations will serve as a reference for evaluating genotype–phenotype relationships.
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the slc37 family of sugar phosphate phosphate exchangers
Current Topics in Membranes, 2014Co-Authors: Janice Y. Chou, Brian C. MansfieldAbstract:The SLC37 family members are endoplasmic reticulum (ER)-associated sugar-phosphate/phosphate (P(i)) exchangers. Three of the four members, SLC37A1, SLC37A2, and SLC37A4, function as Pi-linked glucose-6-phosphate (G6P) antiporters catalyzing G6P:P(i) and P(i):P(i) exchanges. The activity of SLC37A3 is unknown. SLC37A4, better known as the G6P transporter (G6PT), has been extensively characterized, functionally and structurally, and is the best characterized family member. G6PT contains 10 transmembrane helices with both N and C termini facing the cytoplasm. The primary in vivo function of the G6PT protein is to translocate G6P from the cytoplasm into the ER lumen where it couples with either the liver/kidney/intestine-restricted glucose-6-phosphatase-α (G6Pase-α or G6PC) or the ubiquitously expressed G6Pase-β (or G6PC3) to hydrolyze G6P to glucose and P(i). The G6PT/G6Pase-α complex maintains interprandial glucose homeostasis, and the G6PT/G6Pase-β complex maintains neutrophil energy homeostasis and functionality. G6PT is highly selective for G6P and is competitively inhibited by cholorogenic acid and its derivatives. Neither SLC37A1 nor SLC37A2 can couple functionally with G6Pase-α or G6Pase-β, and the antiporter activities of SLC37A1 or SLC37A2 are not inhibited by cholorogenic acid. Deficiencies in G6PT cause glycogen storage disease type Ib (GSD-Ib), a metabolic and immune disorder. To date, 91 separate SLC37A4 mutations, including 39 missense mutations, have been identified in GSD-Ib patients. Characterization of missense mutations has yielded valuable information on functionally important residues in the G6PT protein. The biological roles of the other SLC37 proteins remain to be determined and deficiencies have not yet been correlated to diseases.
Jorge Filmus - One of the best experts on this subject based on the ideXlab platform.
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processing by convertases is required for glypican 3 induced inhibition of hedgehog signaling
Journal of Biological Chemistry, 2015Co-Authors: Mariana I Capurro, Wen Shi, Tomomi Izumikawa, Hiroshi Kitagawa, Jorge FilmusAbstract:Abstract Glypican-3 (GPC3) is one of the six members of the mammalian glypican family. We have previously reported that GPC3 inhibits Hedgehog (Hh) signaling by competing with Patched (Ptc) for Hh binding. We also showed that GPC3 binds with high affinity to Hh through its core protein, but that it does not interact with Ptc. Several members of the glypican family, including GPC3, are subjected to an endoproteolytic cleavage by the furin-like convertase family of endoproteases. Surprisingly, however, we have found that a mutant GPC3 that cannot be processed by convertases is as potent as wild-type GPC3 in stimulating Wnt activity in HCC cell lines and 293T cells, and in promoting HCC growth. In this study we show that processing by convertases is essential for GPC3-induced inhibition of Hh signaling. Moreover, we show that a convertase-resistant GPC3 stimulates Hh signaling by increasing the binding of this growth factor to Ptc. Consistent with this, we show that the convertase-resistant mutant binds to both Hh and Ptc through its heparan sulfate (HS) chains. Unexpectedly, we found that the mutant core protein does not bind to Hh. We also report that the convertase-resistant mutant GPC3 carries HS chains with a significantly higher degree of sulfation than those of wild-type GPC3. We propose that the structural changes generated by the lack of cleavage determine a change in the sulfation of the HS chains, and that these hypersulfated chains mediate the interaction of the mutant GPC3 with Ptc.
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overgrowth of a mouse model of simpson golabi behmel syndrome is partly mediated by indian hedgehog
EMBO Reports, 2009Co-Authors: Mariana I Capurro, Jorge FilmusAbstract:Loss-of-function mutations of Glypican 3 (Gpc3) cause the Simpson–Golabi–Behmel overgrowth syndrome (SGBS), and developmental overgrowth is observed in Gpc3-null mice, a mouse model for SGBS. We recently reported that GPC3 inhibits Hedgehog (Hh) signalling by inducing its endocytosis and degradation. Here, we show that the developmental overgrowth observed in Gpc3-null mice is, at least in part, a consequence of the hyperactivation of the Hh pathway. We bred Gpc3-null mice with mice that are Hh signalling-deficient owing to the lack of Indian Hh (Ihh), one of the three mammalian Hhs. We found that the Gpc3-null mice showed a 29.9% overgrowth in an Ihh wild-type background, whereas an Ihh-null background partly rescues the overgrowth caused by the lack of Gpc3 as the double mutants were 19.8% bigger than the Ihh-null mice. Consistent with the role of GPC3 in Hh endocytosis and degradation, the Gpc3-null mice show increased levels of Ihh protein and signalling, but similar levels of Ihh messenger RNA.
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Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation
Breast Cancer Research and Treatment, 2009Co-Authors: Ivan Stigliano, Jorge Filmus, Lydia Puricelli, Mari Cleide Sogayar, Elisa Bal De Kier Joffé, María Giselle PetersAbstract:Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.
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glypican 3 inhibits hedgehog signaling during development by competing with patched for hedgehog binding
Developmental Cell, 2008Co-Authors: Mariana I Capurro, Wen Shi, Angela Jia, Jorge FilmusAbstract:Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, we hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. Here, we show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, we report that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. We conclude that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway.
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glypican 3 promotes the growth of hepatocellular carcinoma by stimulating canonical wnt signaling
Cancer Research, 2005Co-Authors: Mariana I Capurro, Yunyan Xiang, Corrinne G Lobe, Jorge FilmusAbstract:Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is bound to the cell membrane by a glycosyl-phosphatidylinositol anchor. GPC3 is expressed by most hepatocellular carcinomas but not by normal hepatocytes and benign liver lesions. We report here that GPC3 stimulates the in vitro and in vivo growth of hepatocellular carcinoma cells by increasing autocrine/paracrine canonical Wnt signaling. Coimmunoprecipitation experiments showed that GPC3 is able to form complexes with Wnts, and cell-binding assays indicated that GPC3-expressing cells have an increased capacity to bind Wnt. Collectively, these results suggest that GPC3 stimulates Wnt activity by facilitating the interaction of this polypeptide with its signaling receptors. Surprisingly, in contrast to the current model that proposes that Wnt-glypican binding is mediated by the heparan sulfate chains, we found that the nonglycanated GPC3 core protein can form complexes with Wnts. Furthermore, we showed that the glycosaminoglycan chains are not required for the stimulatory effect on Wnt signaling and hepatocellular carcinoma growth.
Richard M Obrien - One of the best experts on this subject based on the ideXlab platform.
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functional analysis of mouse g6pc1 mutations using a novel in situ assay for glucose 6 phosphatase activity and the effect of mutations in conserved human g6pc1 g6pc2 amino acids on g6pc2 protein expression
PLOS ONE, 2016Co-Authors: Kayla A Boortz, Kristen E Syring, Lynley D Pound, Yingda Wang, James K Oeser, Richard M ObrienAbstract:Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.
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glucose 6 phosphatase catalytic subunit gene family
Journal of Biological Chemistry, 2009Co-Authors: John C Hutton, Richard M ObrienAbstract:Glucose-6-phosphatase catalyzes the hydrolysis of glucose 6-phosphate (G6P) to glucose and inorganic phosphate. It is a multicomponent system located in the endoplasmic reticulum that comprises several integral membrane proteins, namely a catalytic subunit (G6PC) and transporters for G6P, inorganic phosphate, and glucose. The G6PC gene family contains three members, designated G6PC, G6PC2, and G6PC3. The tissue-specific expression patterns of these genes differ, and mutations in all three genes have been linked to distinct diseases in humans. This minireview discusses the disease association and transcriptional regulation of the G6PC genes as well as the biological functions of the encoded proteins.
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deletion of the gene encoding the islet specific glucose 6 phosphatase catalytic subunit related protein autoantigen results in a mild metabolic phenotype
Diabetologia, 2007Co-Authors: Yingda Wang, John C Hutton, James K Oeser, Cyrus C Martin, Suparna A Sarkar, Owen P Mcguinness, Richard M ObrienAbstract:Aims/hypothesis Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, now known as glucose-6-phosphatase, catalytic, 2 [G6PC2]) has recently been identified as a major autoantigen in mouse and human type 1 diabetes. Strategies designed to suppress expression of the gene encoding G6PC2 might therefore be useful in delaying or preventing the onset of this disease. However, since the function of G6PC2 is unclear, the concern with such an approach is that a change in G6PC2 expression might itself have deleterious consequences.
Janice Y. Chou - One of the best experts on this subject based on the ideXlab platform.
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gene therapy using a novel g6pc s298c variant enhances the long term efficacy for treating glycogen storage disease type ia
Biochemical and Biophysical Research Communications, 2020Co-Authors: Lisa Zhang, Brian C. Mansfield, Javier Anduaga, Matthew F Starost, Cheol Woo Lee, Irina Arnaoutova, Janice Y. ChouAbstract:Abstract The current phase I/II clinical trial for human glycogen storage disease type-Ia (GSD-Ia) (NCT 03517085) uses a recombinant adeno-associated virus (rAAV) vector expressing a codon-optimized human glucose-6-phosphatase-α (G6Pase-α or G6PC). DNA sequence changes introduced by codon-optimization can negatively impact gene expression. We therefore generated a novel variant in which a single amino acid change, S298C, is introduced into the native human G6PC sequence. Short term gene transfer study in G6pc−/− mice showed that the rAAV-G6PC-S298C vector is 3-fold more efficacious than the native rAAV-G6PC vector. We have shown previously that restoring 3% of normal hepatic G6Pase-α activity in G6pc−/− mice prevents hepatocellular adenoma/carcinoma (HCA/HCC) development and that mice harboring
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glycogen storage disease type ia mice with less than 2 of normal hepatic glucose 6 phosphatase α activity restored are at risk of developing hepatic tumors
Molecular Genetics and Metabolism, 2017Co-Authors: Goo-young Kim, Brian C. Mansfield, Joonhyun Kwon, Jun-ho Cho, Young Mok Lee, Matthew F Starost, Chijiunn Pan, Janice Y. ChouAbstract:Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA) and carcinoma (HCC), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). We have previously shown that G6pc-/- mice receiving gene transfer mediated by rAAV-G6PC, a recombinant adeno-associated virus (rAAV) vector expressing G6Pase-α, and expressing 3-63% of normal hepatic G6Pase-α activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-α activity required to prevent tumor formation remained unknown. In this study, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-α and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-α expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3-33% of normal hepatic G6Pase-α activity (AAV mice) maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-α activity (AAV-low mice), 3 expressing 0.9-1.3% of normal hepatic G6Pase-α activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing ≥3% of normal hepatic G6Pase-α activity. The results establish the threshold of hepatic G6Pase-α activity required to prevent HCA/HCC and show that GSD-Ia mice harboring <2% of normal hepatic G6Pase-α activity are at risk of tumor development.
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type i glycogen storage diseases disorders of the glucose 6 phosphatase glucose 6 phosphate transporter complexes
Journal of Inherited Metabolic Disease, 2015Co-Authors: Janice Y. Chou, Brian C. Mansfield, Hyun Sik JunAbstract:Disorders of the glucose-6-phosphatase (G6Pase)/glucose-6-phosphate transporter (G6PT) complexes consist of three subtypes: glycogen storage disease type Ia (GSD-Ia), deficient in the liver/kidney/intestine-restricted G6Pase-α (or G6PC); GSD-Ib, deficient in a ubiquitously expressed G6PT (or SLC37A4); and G6Pase-β deficiency or severe congenital neutropenia syndrome type 4 (SCN4), deficient in the ubiquitously expressed G6Pase-β (or G6PC3). G6Pase-α and G6Pase-β are glucose-6-phosphate (G6P) hydrolases with active sites lying inside the endoplasmic reticulum (ER) lumen and as such are dependent upon the G6PT to translocate G6P from the cytoplasm into the lumen. The tissue expression profiles of the G6Pase enzymes dictate the disease's phenotype. A functional G6Pase-α/G6PT complex maintains interprandial glucose homeostasis, while a functional G6Pase-β/G6PT complex maintains neutrophil/macrophage energy homeostasis and functionality. G6Pase-β deficiency is not a glycogen storage disease but biochemically it is a GSD-I related syndrome (GSD-Irs). GSD-Ia and GSD-Ib patients manifest a common metabolic phenotype of impaired blood glucose homeostasis not shared by GSD-Irs. GSD-Ib and GSD-Irs patients manifest a common myeloid phenotype of neutropenia and neutrophil/macrophage dysfunction not shared by GSD-Ia. While a disruption of the activity of the G6Pase-α/G6PT complex readily explains why GSD-Ia and GSD-Ib patients exhibit impaired glucose homeostasis, the basis for neutropenia and myeloid dysfunction in GSD-Ib and GSD-Irs are only now starting to be understood. Animal models of all three disorders are now available and are being exploited to both delineate the disease more precisely and develop new treatment approaches, including gene therapy.
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mesenchymal stem cells lacking glucose 6 phosphatase β exhibit disturbed energy homeostasis and defective adipogenesis
Blood, 2014Co-Authors: Hyun Sik Jun, Janice Y. ChouAbstract:Glucose-6-phosphatase-β (G6Pase-β or G6PC3) is a ubiquitously expressed glucose-6-phosphate (G6P) hydrolase that catalyzes the hydrolysis of G6P to glucose and phosphate. It is a key enzyme for intracellular glucose production in non-gluconeogenic organs. Mutations in G6Pase-β underlie severe congenital neutropenia syndrome type 4 (SCN4, MIM612541), an autosomal recessive disease characterized by neutropenia and neutrophil dysfunction. A mouse model for G6Pase-β deficiency ( G6PC3 -/- ) established by gene targeting manifests both neutropenia and neutrophil dysfunction, mimicking the human disorder. Recent studies have shown that in G6Pase-β deficiency enhanced neutrophil apoptosis leads to neutropenia while disrupted energy homeostasis underlies neutrophil dysfunction. The patients of G6Pase-β deficiency also present with non-haematological defects, including prominent superficial venous pattern, congenital cardiac anomaly, myopathy, disrupted bone remodeling, and genital anomalies, suggesting that G6Pase-β deficiency leads to a broader cell dysfunction. We now hypothesize that these non-haematological defects may reflect loss of glucose metabolism within mesenchymal stem cells (MSC) that require levels of glucose beyond those supplied by the blood. MSC are multipotent stem cells that are capable of differentiating into all connective tissue types including bone, cartilage, myocytes, and adipocytes. Studies have shown that MSC cultured in reduced glucose concentrations exhibit increased proliferative ability and reduced replicative senescence. Using MSC isolated from the compact bones, we show that G6PC3 -/- MSC grow at a significantly faster rate than those of wild type MSC under both normoxia and hypoxia conditions. Consistently, the colony-forming unit-fibroblastic counts in G6PC3 -/- MSC are 2-fold higher than those in wild type MSC, suggesting that G6PC3 -/- MSC contain higher numbers of progenitors. Differentiation of MSC involves metabolic shifts between glycolysis and mitochondrial oxidative phosphorylation. An increase in the ratio of lactate to pyruvate correlates with increased glycolysis and an increase in oxygen consumption measures increased mitochondrial oxidative phosphorylation. Adipogenesis and osteogenesis of MSC have been shown to be associated with increased oxygen consumption, while chondrogenesis of MSC is associated with increased glycolysis. We now show that cellular levels of lactate and ATP in G6PC3 -/- MSC are 4.5- and 2-fold higher, respectively than those in wide type MSC. Moreover, G6PC3 -/- MSC exhibit a 2.5-fold increase in the ratio of lactate to pyruvate but a decrease in oxygen consumption, compared to wild type MSC, suggesting that G6Pase-β-deficient MSC exhibit enhanced glycolysis along with impaired mitochondrial respiration activity. This also suggests that adipogenesis and osteogenesis of MSC may be impaired in G6PC3 -/- MSC. Adipogenic differentiation of MSC from mice was then evaluated by morphological alterations and lipid accumulation. MSC from wild type mice, cultured for 3 days in adipogenic differentiation medium, changed from fibroblast-like cells to round-shaped adipocyte-like cells, while G6PC3 -/- MSC cultured under the same conditions did not show any morphological changes. Wild type MSC, cultured for 5 days in adipogenic differentiation medium, yielded many adipocytes containing lipid droplets and staining positive by Oil red O, while adipocytes were rarely seen in G6PC3 -/- MSC cultured under similar conditions, confirming the impaired adipogenic differentiation of G6PC3 -/- MSC. In conclusion, the non-hematological defects associated with G6Pase-β deficiency may result from altered energy homeostasis in G6PC3 -/- MSC, leading to enhanced glycolysis along with impaired mitochondrial respiration activity, that impacts MSC differentiation. Disclosures No relevant conflicts of interest to declare.
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the slc37 family of sugar phosphate phosphate exchangers
Current Topics in Membranes, 2014Co-Authors: Janice Y. Chou, Brian C. MansfieldAbstract:The SLC37 family members are endoplasmic reticulum (ER)-associated sugar-phosphate/phosphate (P(i)) exchangers. Three of the four members, SLC37A1, SLC37A2, and SLC37A4, function as Pi-linked glucose-6-phosphate (G6P) antiporters catalyzing G6P:P(i) and P(i):P(i) exchanges. The activity of SLC37A3 is unknown. SLC37A4, better known as the G6P transporter (G6PT), has been extensively characterized, functionally and structurally, and is the best characterized family member. G6PT contains 10 transmembrane helices with both N and C termini facing the cytoplasm. The primary in vivo function of the G6PT protein is to translocate G6P from the cytoplasm into the ER lumen where it couples with either the liver/kidney/intestine-restricted glucose-6-phosphatase-α (G6Pase-α or G6PC) or the ubiquitously expressed G6Pase-β (or G6PC3) to hydrolyze G6P to glucose and P(i). The G6PT/G6Pase-α complex maintains interprandial glucose homeostasis, and the G6PT/G6Pase-β complex maintains neutrophil energy homeostasis and functionality. G6PT is highly selective for G6P and is competitively inhibited by cholorogenic acid and its derivatives. Neither SLC37A1 nor SLC37A2 can couple functionally with G6Pase-α or G6Pase-β, and the antiporter activities of SLC37A1 or SLC37A2 are not inhibited by cholorogenic acid. Deficiencies in G6PT cause glycogen storage disease type Ib (GSD-Ib), a metabolic and immune disorder. To date, 91 separate SLC37A4 mutations, including 39 missense mutations, have been identified in GSD-Ib patients. Characterization of missense mutations has yielded valuable information on functionally important residues in the G6PT protein. The biological roles of the other SLC37 proteins remain to be determined and deficiencies have not yet been correlated to diseases.
Siddharth Banka - One of the best experts on this subject based on the ideXlab platform.
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a clinical and molecular review of ubiquitous glucose 6 phosphatase deficiency caused by G6PC3 mutations
Orphanet Journal of Rare Diseases, 2013Co-Authors: Siddharth Banka, William G NewmanAbstract:The G6PC3 gene encodes the ubiquitously expressed glucose-6-phosphatase enzyme (G-6-Pase β or G-6-Pase 3 or G6PC3). Bi-allelic G6PC3 mutations cause a multi-system autosomal recessive disorder of G6PC3 deficiency (also called severe congenital neutropenia type 4, MIM 612541). To date, at least 57 patients with G6PC3 deficiency have been described in the literature. G6PC3 deficiency is characterized by severe congenital neutropenia, recurrent bacterial infections, intermittent thrombocytopenia in many patients, a prominent superficial venous pattern and a high incidence of congenital cardiac defects and uro-genital anomalies. The phenotypic spectrum of the condition is wide and includes rare manifestations such as maturation arrest of the myeloid lineage, a normocellular bone marrow, myelokathexis, lymphopaenia, thymic hypoplasia, inflammatory bowel disease, primary pulmonary hypertension, endocrine abnormalities, growth retardation, minor facial dysmorphism, skeletal and integument anomalies amongst others. Dursun syndrome is part of this extended spectrum. G6PC3 deficiency can also result in isolated non-syndromic severe neutropenia. G6PC3 mutations in result in reduced enzyme activity, endoplasmic reticulum stress response, increased rates of apoptosis of affected cells and dysfunction of neutrophil activity. In this review we demonstrate that loss of function in missense G6PC3 mutations likely results from decreased enzyme stability. The condition can be diagnosed by sequencing the G6PC3 gene. A number of G6PC3 founder mutations are known in various populations and a possible genotype-phenotype relationship also exists. G6PC3 deficiency should be considered as part of the differential diagnoses in any patient with unexplained congenital neutropenia. Treatment with G-CSF leads to improvement in neutrophil numbers, prevents infections and improves quality of life. Mildly affected patients can be managed with prophylactic antibiotics. Untreated G6PC3 deficiency can be fatal. Echocardiogram, renal and pelvic ultrasound scans should be performed in all cases of suspected or confirmed G6PC3 deficiency. Routine assessment should include biochemical profile, growth profile and monitoring for development of varicose veins or venous ulcers.
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G6PC3 mutations cause non syndromic severe congenital neutropenia
Molecular Genetics and Metabolism, 2013Co-Authors: Siddharth Banka, Robert Wynn, Helen Byers, Peter D Arkwright, William G NewmanAbstract:The deficiency of ubiquitously expressed glucose-6-phosphatase (G6PC3) enzyme is known to result in a syndrome characterized by severe congenital neutropenia, prominent superficial venous pattern, congenital heart defects and genito-urinary malformations. Here, we describe four patients from three families with non-syndromic severe congenital neutropenia and identify four G6PC3 mutations as causative in these cases. Thus we demonstrate that G6PC3 mutations also result in a non-syndromic form of severe congenital neutropenia. We propose that G6PC3 deficiency should be considered as part of the differential diagnoses in any patient with unexplained congenital neutropenia. Additionally, we show a relationship between the genotype and non-hematological phenotype of G6PC3 deficiency. These findings may provide an insight into the role of the G6PC3 enzyme and glucose metabolism in developmental pathways.
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extended spectrum of human glucose 6 phosphatase catalytic subunit 3 deficiency novel genotypes and phenotypic variability in severe congenital neutropenia
The Journal of Pediatrics, 2012Co-Authors: Kaan Boztug, Siddharth Banka, Philip S Rosenberg, Marie Dorda, Thomas Moulton, Julie Curtin, Nima Rezaei, John CornsAbstract:OBJECTIVE: To delineate the phenotypic and molecular spectrum of patients with a syndromic variant of severe congenital neutropenia (SCN) due to mutations in the gene encoding glucose-6-phosphatase catalytic subunit 3 (G6PC3). STUDY DESIGN: Patients with syndromic SCN were characterized for associated malformations and referred to us for G6PC3 mutational analysis. RESULTS: In a cohort of 31 patients with syndromic SCN, we identified 16 patients with G6PC3 deficiency including 11 patients with novel biallelic mutations. We show that nonhematologic features of G6PC3 deficiency are good predictive indicators for mutations in G6PC3. Additionally, we demonstrate genetic variability in this disease and define novel features such as growth hormone deficiency, genital malformations, disrupted bone remodeling, and abnormalities of the integument. G6PC3 mutations may be associated with hydronephrosis or facial dysmorphism. The risk of transition to myelodysplastic syndrome/acute myeloid leukemia may be lower than in other genetically defined SCN subgroups. CONCLUSIONS: The phenotypic and molecular spectrum in G6PC3 deficiency is wider than previously appreciated. The risk of transition to myelodysplastic syndrome or acute myeloid leukemia may be lower in G6PC3 deficiency compared with other subgroups of SCN.
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mutations in the G6PC3 gene cause dursun syndrome
American Journal of Medical Genetics Part A, 2010Co-Authors: Siddharth Banka, William G Newman, Koksal R Ozgul, Ali DursunAbstract:Dursun syndrome is a triad of familial primary pulmonary hypertension, leucopenia, and atrial septal defect. Here we demonstrate that mutations in G6PC3 cause Dursun syndrome. Mutations in G6PC3 are known to also cause severe congenital neutropenia type 4. Identification of the genetic basis of Dursun syndrome expands the pre-existing knowledge about the phenotypic effects of mutations in G6PC3. We propose that Dursun syndrome should now be considered as a subset of severe congenital neutropenia type 4 with pulmonary hypertension as an important clinical feature.
