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Glycogen 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-alpha (G6Pase-alpha) or by a ubiquitously expressed G6Pase-beta. The role of the G6PT/G6Pase-alpha 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.Characterization of G6Pase-beta and generation of mice lacking either G6PT or G6Pase-beta have shown that neutrophils express the G6PT/G6Pase-beta 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.Neutrophil function is intimately linked to the regulation of glucose and G6P metabolism by the G6PT/G6Pase-beta complex. Understanding the molecular mechanisms that govern energy homeostasis in neutrophils has revealed a previously unrecognized pathway of intracellular G6P metabolism in neutrophils.

The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes , a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to β-1→4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased k cat /K m values for cello-oligosaccharides with higher degrees of polymerization. The k cat /K m value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-β- d -glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.

The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. succinogenes , Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.

Women's health has been threatened by various diseases mainly including heart disease, breast cancer, osteoporosis, depression, and autoimmune disease. But development of medication for these diseases has been restricted by high development costs and low success rates. Herein the attempt to develop valid bioactive materials from a traditional natural material has been made. Resveratrol has been reported to be effective in treatment of breast cancer and heart disease. Goji berry has received attention as a natural based therapeutic material to treat a diabetes, cardiovascular disease, and osteoporosis. Leonurus family has been reported to be effective particularly in pregnant women due to high contents of vitamin as well as stimulation of uterine contraction. Annona family has effects such as anti-anxiety, anticonvulsant and recently it is proposed to be as a therapeutic material to cure depression based on its strong antidepressant effect. Shiraia bambusicola has been utilized to cure angiogenesis-related disease from ancient China and furthermore recently it was proved to be effective in rheumatoid arthritis. Getting an understanding of utilization of these traditional natural materials not only enhances the interest in development of therapeutic materials for preventing and treating various women's diseases, but also makes it possible to develop novel therapeutic materials.

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.

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.

Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER) of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P) into glucose and phosphate (P(i)). This reaction depends on coupling the G6P transporter (G6PT) with glucose-6-phosphatase-α (G6Pase-α). Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a P(i)-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:P(i) exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, P(i)-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.