Brian C. Mansfield

Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse

Glycogen storage disease type 1a (GSD–1a) is caused by a deficiency in microsomal glucose–6–phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD–1 a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose–6–P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis

Glycogen storage disease type I and G6Pase-β deficiency: etiology and therapy

Glycogen storage disease type I (GSD-I) consists of two subtypes: GSD-Ia, a deficiency in glucose-6-phosphatase-α (G6Pase-α) and GSD-Ib, which is characterized by an absence of a glucose-6-phosphate (G6P) transporter (G6PT). A third disorder, G6Pase-β deficiency, shares similarities with this group of diseases. G6Pase-α and G6Pase-β are G6P hydrolases in the membrane of the endoplasmic reticulum, which depend on G6PT to transport G6P from the cytoplasm into the lumen. A functional complex of G6PT and G6Pase-α maintains interprandial glucose homeostasis, whereas G6PT and G6Pase-β act in conjunction to maintain neutrophil function and homeostasis. Patients with GSD-Ia and those with GSD-Ib exhibit a common metabolic phenotype of disturbed glucose homeostasis that is not evident in patients with G6Pase-β deficiency. Patients with a deficiency in G6PT and those lacking G6Pase-β display a common myeloid phenotype that is not shared by patients with GSD-Ia. Previous studies have shown that neutrophils express the complex of G6PT and G6Pase-β to produce endogenous glucose. Inactivation of either G6PT or G6Pase-β increases neutrophil apoptosis, which underlies, at least in part, neutrophil loss (neutropenia) and dysfunction in GSD-Ib and G6Pase-β deficiency. Dietary and/or granulocyte colony-stimulating factor therapies are available; however, many aspects of the diseases are still poorly understood. This Review will address the etiology of GSD-Ia, GSD-Ib and G6Pase-β deficiency and highlight advances in diagnosis and new treatment approaches, including gene therapy.

Impaired neutrophil activity and increased susceptibility to bacterial infection in mice lacking glucose-6-phosphatase–β

Neutropenia and neutrophil dysfunction are common in many diseases, although their etiology is often unclear. Previous views held that there was a single ER enzyme, glucose-6-phosphatase-alpha (G6Pase-alpha), whose activity--limited to the liver, kidney, and intestine--was solely responsible for the final stages of gluconeogenesis and glycogenolysis, in which glucose-6-phosphate (G6P) is hydrolyzed to glucose for release to the blood. Recently, we characterized a second G6Pase activity, that of G6Pase-beta (also known as G6PC), which is also capable of hydrolyzing G6P to glucose but is ubiquitously expressed and not implicated in interprandial blood glucose homeostasis. We now report that the absence of G6Pase-beta led to neutropenia; defects in neutrophil respiratory burst, chemotaxis, and calcium flux; and increased susceptibility to bacterial infection. Consistent with this, G6Pase-beta-deficient (G6pc3-/-) mice with experimental peritonitis exhibited increased expression of the glucose-regulated proteins upregulated during ER stress in their neutrophils and bone marrow, and the G6pc3-/- neutrophils exhibited an enhanced rate of apoptosis. Our results define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology, providing a potential model for the treatment of these conditions.

Mutations in the glucose‐6‐phosphatase‐α (G6PC) gene that cause type Ia glycogen storage disease

Glucose‐6‐phosphatase‐α (G6PC) is a key enzyme in glucose homeostasis that catalyzes the hydrolysis of glucose‐6‐phosphate to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis. Mutations in the G6PC gene, located on chromosome 17q21, result in glycogen storage disease type Ia (GSD‐Ia), an autosomal recessive metabolic disorder. GSD‐Ia patients manifest a disturbed glucose homeostasis, characterized by fasting hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, lactic acidemia, and growth retardation. G6PC is a highly hydrophobic glycoprotein, anchored in the membrane of the endoplasmic reticulum with the active center facing into the lumen. To date, 54 missense, 10 nonsense, 17 insertion/deletion, and three splicing mutations in the G6PC gene have been identified in more than 550 patients. Of these, 50 missense, two nonsense, and two insertion/deletion mutations have been functionally characterized for their effects on enzymatic activity and stability. While GSD‐Ia is not more prevalent in any ethnic group, mutations unique to Caucasian, Oriental, and Jewish populations have been described. Despite this, GSD‐Ia patients exhibit phenotypic heterogeneity and a stringent genotype–phenotype relationship does not exist. Hum Mutat 29(7), 921–930, 2008. Published 2008 Wiley‐Liss, Inc.

Molecular Genetics of Type 1 Glycogen Storage Diseases.

Glycogen storage disease type 1 (GSD-1), also known as von Gierke disease, is caused by a deficiency in the activity of the enzyme glucose-6-phosphatase (G6Pase). It is an autosomal recessive disorder characterized by hypoglycemia, hepatomegaly, kidney enlargement, growth retardation, lactic acidemia, hyperlipidemia and hyperuricemia. The disease presents with both clinical and biochemical heterogeneity consistent with the existence of two major subgroups, GSD-1a and GSD-1b, which have been confirmed at the molecular genetic level. GSD-1a, the most prevalent form, is caused by mutations in the G6Pase gene that abolish or greatly reduce enzymatic activity. The gene maps to chromosome 17q21 and encodes a microsomal transmembrane protein. Animal models of GSD-1a exist and are being exploited to delineate the disease more precisely. It has been proposed that GSD-1b is caused by a defect in the microsomal glucose-6-phosphate transporter. The gene responsible for GSD-1b has been mapped to chromosome 11q23 and a cDNA encoding a microsomal transmembrane protein has been identified. The function of this putative GSD-1b protein remains to be determined. These recent developments, along with newly characterized animal models of GSD-1a, are increasing our understanding of the interrelationship between the components of the G6Pase complex and type 1 glycogen storage diseases.

Development and Preliminary Evaluation of a Multivariate Index Assay for Ovarian Cancer

Background Most women with a clinical presentation consistent with ovarian cancer have benign conditions. Therefore methods to distinguish women with ovarian cancer from those with benign conditions would be beneficial. We describe the development and preliminary evaluation of a serum-based multivariate assay for ovarian cancer. This hypothesis-driven study examined whether an informative pattern could be detected in stage I disease that persists through later stages. Methodology/Principal Findings Sera, collected under uniform protocols from multiple institutions, representing 176 cases and 187 controls from women presenting for surgery were examined using high-throughput, multiplexed immunoassays. All stages and common subtypes of epithelial ovarian cancer, and the most common benign ovarian conditions were represented. A panel of 104 antigens, 44 autoimmune and 56 infectious disease markers were assayed and informative combinations identified. Using a training set of 91 stage I data sets, representing 61 individual samples, and an equivalent number of controls, an 11-analyte profile, composed of CA-125, CA 19-9, EGF-R, C-reactive protein, myoglobin, apolipoprotein A1, apolipoprotein CIII, MIP-1α, IL-6, IL-18 and tenascin C was identified and appears informative for all stages and common subtypes of ovarian cancer. Using a testing set of 245 samples, approximately twice the size of the model building set, the classifier had 91.3% sensitivity and 88.5% specificity. While these preliminary results are promising, further refinement and extensive validation of the classifier in a clinical trial is necessary to determine if the test has clinical value. Conclusions/Significance We describe a blood-based assay using 11 analytes that can distinguish women with ovarian cancer from those with benign conditions. Preliminary evaluation of the classifier suggests it has the potential to offer approximately 90% sensitivity and 90% specificity. While promising, the performance needs to be assessed in a blinded clinical validation study.

Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α), a nine-transmembrane domain, endoplasmic reticulum-associated protein expressed primarily in the liver and kidney. Previously, we showed that infusion of an adeno-associated virus (AAV) serotype 2 vector carrying murine G6Pase-α (AAV2-G6Pase-α) into neonatal GSD-Ia mice failed to sustain their life beyond weaning. We now show that neonatal infusion of GSD-Ia mice with an AAV serotype 1-G6Pase-α (AAV1-G6Pase-α) or AAV serotype 8-G6Pase-α (AAV8-G6Pase-α) results in hepatic expression of the G6Pase-α transgene and markedly improves the survival of the mice. However, only AAV1-G6Pase-α can achieve significant renal transgene expression. A more effective strategy, in which a neonatal AAV1-G6Pase-α infusion is followed by a second infusion at age one week, provides sustained expression of a complete, functional, G6Pase-α system in both the liver and kidney and corrects the metabolic abnormalities in GSD-Ia mice for the 57 week length of the study. This effective use of gene therapy to correct metabolic imbalances and disease progression in GSD-Ia mice holds promise for the future of gene therapy in humans.

Neutrophil stress and apoptosis underlie myeloid dysfunction in glycogen storage disease type Ib

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that works with a liver/kidney/intestine-restricted glucose-6-phosphatase-alpha (G6Pase-alpha) to maintain glucose homeostasis between meals. Clinically, GSD-Ib patients manifest disturbed glucose homeostasis and neutrophil dysfunctions but the cause of the latter is unclear. Neutrophils express the ubiquitously expressed G6PT and G6Pase-beta that together transport G6P into the endoplasmic reticulum (ER) lumen and hydrolyze it to glucose. Because we expected G6PT-deficient neutrophils to be unable to produce endogenous glucose, we hypothesized this would lead to ER stress and increased apoptosis. Using GSD-Ib mice, we showed that GSD-Ib neutrophils exhibited increased production of ER chaperones and oxidative stress, consistent with ER stress, increased annexin V binding and caspase-3 activation, consistent with an increased rate of apoptosis. Bax activation, mitochondrial release of proapoptotic effectors, and caspase-9 activation demonstrated 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.

Complete Normalization of Hepatic G6PC Deficiency in Murine Glycogen Storage Disease Type Ia Using Gene Therapy

Glycogen storage disease type Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-alpha (G6Pase-alpha or G6PC) manifest disturbed glucose homeostasis. We examined the efficacy of liver G6Pase-alpha delivery mediated by AAV-GPE, an adeno-associated virus (AAV) serotype 8 vector expressing human G6Pase-alpha directed by the human G6PC promoter/enhancer (GPE), and compared it to AAV-CBA, that directed murine G6Pase-alpha expression using a hybrid chicken beta-actin (CBA) promoter/cytomegalovirus (CMV) enhancer. The AAV-GPE directed hepatic G6Pase-alpha expression in the infused G6pc(-/-) mice declined 12-fold from age 2 to 6 weeks but stabilized at wild-type levels from age 6 to 24 weeks. In contrast, the expression directed by AAV-CBA declined 95-fold over 24 weeks, demonstrating that the GPE is more effective in directing persistent in vivo hepatic transgene expression. We further show that the rapid decline in transgene expression directed by AAV-CBA results from an inflammatory immune response elicited by the AAV-CBA vector. The AAV-GPE-treated G6pc(-/-) mice exhibit normal levels of blood glucose, blood metabolites, hepatic glycogen, and hepatic fat. Moreover, the mice maintained normal blood glucose levels even after 6 hours of fasting. The complete normalization of hepatic G6Pase-alpha deficiency by the G6PC promoter/enhancer holds promise for the future of gene therapy in human GSD-Ia patients.

Neutropenia in type Ib glycogen storage disease

Purpose of reviewGlycogen storage disease type Ib, characterized by disturbed glucose homeostasis, neutropenia, and neutrophil dysfunction, is caused by a deficiency in a ubiquitously expressed glucose-6-phosphate transporter (G6PT). G6PT translocates glucose-6-phosphate (G6P) from the cytoplasm into the lumen of the endoplasmic reticulum, in which it is hydrolyzed to glucose either by a liver/kidney/intestine-restricted glucose-6-phosphatase-α (G6Pase-α) or by a ubiquitously expressed G6Pase-β. The role of the G6PT/G6Pase-α complex is well established and readily explains why G6PT disruptions disturb interprandial blood glucose homeostasis. However, the basis for neutropenia and neutrophil dysfunction in glycogen storage disease type Ib is poorly understood. Recent studies that are now starting to unveil the mechanisms are presented in this review. Recent findingsCharacterization of G6Pase-β and generation of mice lacking either G6PT or G6Pase-β have shown that neutrophils express the G6PT/G6Pase-β complex capable of producing endogenous glucose. Loss of G6PT activity leads to enhanced endoplasmic reticulum stress, oxidative stress, and apoptosis that underlie neutropenia and neutrophil dysfunction in glycogen storage disease type Ib. SummaryNeutrophil function is intimately linked to the regulation of glucose and G6P metabolism by the G6PT/G6Pase-β complex. Understanding the molecular mechanisms that govern energy homeostasis in neutrophils has revealed a previously unrecognized pathway of intracellular G6P metabolism in neutrophils.