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Objective: Interleukin-6 (IL-6) is a T cell-derived B cell stimulating factor which plays an important role in inflammatory diseases. In this study, the pharmacokinetic properties of LMT-28 including physicochemical property, in vitro liver microsomal stability and an in vivo pharmacokinetic study using BALB/c mice were characterized.Methods: LMT-28 has been synthesized and is being developed as a novel therapeutic IL-6 inhibitor. The physicochemical properties and in vitro pharmacokinetic profiles such as liver microsomal stability and Madin-Darby canine kidney (MDCK) cell permeability assay were examined. For in vivo pharmacokinetic studies, pharmacokinetic parameters using BALB/c mice were calculated.Results: The logarithm of the partition coefficient value (LogP; 3.65) and the apparent permeability coefficient values (Papp; 9.7×10–6 cm/s) showed that LMT-28 possesses a moderate-high cell permeability property across MDCK cell monolayers. The plasma protein binding rate of LMT-28 was 92.4% and mostly bound to serum albumin. The metabolic half-life (t1/2) values of LMT-28 were 15.3 min for rat and 21.9 min for human at the concentration 1 μM. The area under the plasma drug concentration-time curve and Cmax after oral administration (5 mg/kg) of LMT-28 were 302±209 h∙ng/mL and 137±100 ng/mL, respectively.Conclusion: These data suggest that LMT-28 may have good physicochemical and pharmacokinetic properties and may be a novel oral drug candidate as the first synthetic IL-6 inhibitor to ameliorate mammalian inflammation.

Ferritin (FT), a cage-protein composed of 24 subunits, may carry small molecules inside the core of cage. To enhance the potential of FT as a more functional drug carrier, FT genetically-fused with silaffin R5 peptide at N-terminus (R5FT) and silica-enveloped cage protein (SiO2/R5FT) were developed. Doxorubicin (Dox) can be loaded into cage interior by re-assembly of R5FT. The release of Dox loaded in internal cages was retarded by the silica-coated matrix and showed the pH-controlled behavior, in which the release amount is larger in acidic environments than neutral. R5FT provides a biocompatible and eco-friendly manufacturing process for the drug-laden silica nanoparticles over conventional production methods.

The vine stem of Spatholobus suberectus Dunn (SS) is used as a traditional herbal medicine in China. Chinese herbal medicines are well known as natural bioactive compounds that can be used as new medicines, and their antioxidant and anticancer effects have also been reported. This study aimed to examine the anticancer effect of a high-pressure hot-water SS extract on rat C6 glioma cells. The SS extract effectively suppressed the viability and proliferation of C6 glioma cells through an antioxidant effect. Reactive oxygen species (ROS) levels in cancer cells are higher than that in normal cells. If the ROS level falls below that required for the growth of cancer cells, their rapid proliferation and growth can be suppressed. We also measured the induction of mitochondrial membrane depolarization and cell cycle arrest effect caused by the SS extract in C6 glioma cells through a FACS analysis. In addition, we observed an increase in STAT3, p53, E2F1, and p21 mRNA expression and a decrease in Bcl-2 mRNA expression by quantitative PCR. An increase in p21 protein expression of over 83% was observed through western blot analysis. All these data support the fact that the high-pressure hot-water SS extract has the potential to be used for glioma treatment.

Gliomas are one of the most common types of primary brain tumors, characterized by rapid proliferation and infiltration into normal brain tissue. Corncob is the most plentiful byproducts of Zea mays L., of which anti-cancer effect has not been reported. Therefore, we aimed to examine the anti-proliferative effect of a high-pressure hot-water extract of corncob on glioma cells and elucidated the underlying mechanism. The high-pressure hot-water corncob extract contained approximately 94.8 mg/g and 1.82 μg/g of total phenol and catechin, respectively. Glioma cell treated with different concentrations of high-pressure hot-water corncob extract was shown to be suppressed in growth during three days of culture. In parallel, corncob extract reduced the glioma cell viability and induced cell cycle arrest in G0/G1 phase by upregulating the expression level of cyclin-dependent kinase inhibitor p21. Decreased proliferation and viability in glioma cells treated with corncob extract can be attributed to reduced reactive oxygen species (ROS), antiapoptotic Bcl-2 protein, and a lactate transporter monocarboxylate transporter 1 of which levels are higher than those in normal cells. Based on its inhibitory effects on proliferation and viability of C6 glioma cells, a high-pressure hot-water corncob extract has the potential to be used for glioma treatment.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is an inherited autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Neutrophils play an essential role in the defense against invading pathogens. The recruitment of neutrophils towards the inflammation sites in response to inflammatory stimuli is a tightly regulated process involving rolling, adhesion, and transmigration. In this study, we investigated the role of G6PT in neutrophil adhesion and migration using in vivo and in vitro models. We showed that the GSD-Ib (G6pt(−/−)) mice manifested severe neutropenia in both blood and bone marrow, and treating G6pt(−/−) mice with granulocyte colony-stimulating factor (G-CSF) corrected neutropenia. However, upon thioglycolate challenge, neutrophils from both untreated and G-CSF-treated G6pt(−/−)mice exhibited decreased ability to migrate to the peritoneal cavity. In vitro migration and cell adhesion of G6PT-deficient neutrophils were also significantly impaired. Defects in cell migration were not due to enhanced apoptosis or altered fMLP receptor expression. Remarkably, the expression of the β2 integrins CD11a and CD11b, which are critical for cell adhesion, was greatly decreased in G6PT-deficient neutrophils. This study suggests that deficiencies in G6PT cause impairment in neutrophil adhesion and migration via aberrant expression of β2 integrins, and our finding should facilitate the development of novel therapies for GSD-Ib.

The SLC37 family consists of four sugar-phosphate exchangers, A1, A2, A3, and A4, which are anchored in the endoplasmic reticulum (ER) membrane. The best characterized family member is SLC37A4, better known as the glucose-6-phosphate (G6P) transporter (G6PT). SLC37A1, SLC37A2, and G6PT function as phosphate (Pi)-linked G6P antiporters catalyzing G6P:Pi and Pi:Pi exchanges. The activity of SLC37A3 is unknown. G6PT translocates G6P from the cytoplasm into the lumen of the ER where it couples with either glucose-6-phosphatase-α (G6Pase-α) or G6Pase-β to hydrolyze intraluminal G6P to glucose and Pi. The functional coupling of G6PT with G6Pase-α maintains interprandial glucose homeostasis and the functional coupling of G6PT with G6Pase-β maintains neutrophil energy homeostasis and functionality. A deficiency in G6PT causes glycogen storage disease type Ib, an autosomal recessive disorder characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction. Neither SLC37A1 nor SLC37A2 can functionally couple with G6Pase-α or G6Pase-β, and there are no known disease associations for them or SLC37A3. Since only G6PT matches the characteristics of the physiological ER G6P transporter involved in blood glucose homeostasis and neutrophil energy metabolism, the biological roles for the other SLC37 proteins remain to be determined.

Glycogen storage disease type Ia (GSD-Ia) patients, deficient in glucose-6-phosphatase-alpha, manifest disturbed glucose homeostasis with long-term renal disease. We have previously shown that renal fibrosis in GSD-Ia is mediated by the angiotensin/transforming growth factor-beta1 (TGF-beta1) pathway, which also elicits renal damage through oxidative stress. In this study, we further elucidate the mechanism of renal disease by showing that renal expression of Nox-2, p22(phox), and p47(phox), components of NADPH oxidase, are upregulated in GSD-Ia mice compared with controls. Akt/protein kinase B, a downstream mediator of angiotensin II and TGF-beta1, is also activated, leading to phosphorylation and inactivation of the Forkhead box O family of transcription factors. This in turn triggers downregulation of superoxide dismutase and catalase (CAT) activities that have essential roles in oxidative detoxification in mammals. Renal oxidative stress in GSD-Ia mice is shown by increased oxidation of dihydroethidium and by oxidative damage of DNA. Importantly, renal dysfunction, reflected by elevated serum levels of blood urea nitrogen, reduced renal CAT activity, and increased renal fibrosis, is improved in GSD-Ia mice treated with the antioxidant drug tempol. These data provide the first evidence that oxidative stress is one mechanism that underlies GSD-Ia nephropathy.

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-α (G6Pase-α) 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-β 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.

Comparative analysis of binding of intact glucose-grownFibrobacter succinogenesstrain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grownF. succinogeneswere identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system ofF. succinogenes.

Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes.

Glycogen storage disease type Ib (GSD-Ib) is an autosomal-recessive syndrome characterized by neutropenia and impaired glucose homeostasis resulting from a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT). The underlying cause of GSD-Ib neutropenia is an enhanced neutrophil apoptosis, but patients also manifest neutrophil dysfunction of unknown etiology. Previously, we showed G6PT interacts with the enzyme glucose-6-phosphatase-β (G6Pase-β) to regulate the availability of G6P/glucose in neutrophils. A deficiency in G6Pase-β activity in neutrophils impairs both their energy homeostasis and function. We now show that G6PT-deficient neutrophils from GSD-Ib patients are similarly impaired. Their energy impairment is characterized by decreased glucose uptake and reduced levels of intracellular G6P, lactate, adenosine triphosphate, and reduced NAD phosphate, whereas functional impairment is reflected in reduced neutrophil respiratory burst, chemotaxis, and calcium mobilization. We further show that the mechanism of neutrophil dysfunction in GSD-Ib arises from activation of the hypoxia-inducible factor-1α/peroxisome-proliferators–activated receptor-γ pathway.

Glucose-6-phosphatase-β (G6Pase-β or G6PC3) deficiency, also known as severe congenital neutropenia syndrome 4, is characterized not only by neutropenia but also by impaired neutrophil energy homeostasis and functionality. We now show the syndrome is also associated with macrophage dysfunction, with murine G6pc3−/− macrophages having impairments in their respiratory burst, chemotaxis, calcium flux, and phagocytic activities. Consistent with a glucose-6-phosphate (G6P) metabolism deficiency, G6pc3−/− macrophages also have a lower glucose uptake and lower levels of G6P, lactate, and ATP than wild-type macrophages. Furthermore, the expression of NADPH oxidase subunits and membrane translocation of p47phox are down-regulated, and G6pc3−/− macrophages exhibit repressed trafficking in vivo both during an inflammatory response and in pregnancy. During pregnancy, the absence of G6Pase-β activity also leads to impaired energy homeostasis in the uterus and reduced fertility of G6pc3−/− mothers. Together these results show that immune deficiencies in this congenital neutropenia syndrome extend beyond neutrophil dysfunction.

Glycogen storage disease type Ia (GSD-Ia), which is characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA), is caused by deficiencies in the endoplasmic reticulum (ER)-associated glucose-6-phosphatase-α (G6Pase-α or G6PC) that hydrolyzes glucose-6-phosphate (G6P) to glucose. G6Pase-α activity depends on the G6P transporter (G6PT) that translocates G6P from the cytoplasm into the ER lumen. The functional coupling of G6Pase-α and G6PT maintains interprandial glucose homeostasis. We have shown previously that gene therapy mediated by AAV-GPE, an adeno-associated virus (AAV) vector expressing G6Pase-α directed by the human G6PC promoter/enhancer (GPE), completely normalizes hepatic G6Pase-α deficiency in GSD-Ia (G6pc−/−) mice for at least 24 weeks. However, a recent study showed that within 78 weeks of gene deletion, all mice lacking G6Pase-α in the liver develop HCA. We now show that gene therapy mediated by AAV-GPE maintains efficacy for at least 70-90 weeks for mice expressing more than 3% of wild-type hepatic G6Pase-α activity. The treated mice displayed normal hepatic fat storage, had normal blood metabolite and glucose tolerance profiles, had reduced fasting blood insulin levels, maintained normoglycemia over a 24-hour fast, and had no evidence of hepatic abnormalities. After a 24-hour fast, hepatic G6PT messenger RNA levels in G6pc−/− mice receiving gene therapy were markedly increased. Because G6PT transport is the rate-limiting step in microsomal G6P metabolism, this may explain why the treated G6pc−/− mice could sustain prolonged fasts. The low fasting blood insulin levels and lack of hepatic steatosis may explain the absence of HCA. Conclusion: These results confirm that AAV-GPE–mediated gene transfer corrects hepatic G6Pase-α deficiency in murine GSD-Ia and prevents chronic HCA formation. (HEPATOLOGY 2012;56:1719–1729)

G6PC3 (or glucose-6-phosphatase-β) deficiency underlies a congenital neutropenia syndrome in which neutrophils exhibit enhanced endoplasmic reticulum (ER) stress, increased apoptosis, impaired energy homeostasis, and impaired functionality. Here we show that murine G6pc3−/− neutrophils undergoing ER stress activate protein kinase-like ER kinase and phosphatidylinositol 3,4,5-trisphosphate/Akt signaling pathways, and that neutrophil apoptosis is mediated in part by the intrinsic mitochondrial pathway. In G6PC3-deficient patients, granulocyte colony-stimulating factor (G-CSF) improves neutropenia, but its impact on neutrophil apoptosis and dysfunction is unknown. We now show that G-CSF delays neutrophil apoptosis in vitro by modulating apoptotic mediators. However, G6pc3−/− neutrophils in culture exhibit accelerated apoptosis compared with wild-type neutrophils both in the presence or absence of G-CSF. Limiting glucose (0.6mM) accelerates apoptosis but is more pronounced for wild-type neutrophils, leading to similar survival profiles for both neutrophil populations. In vivo G-CSF therapy completely corrects neutropenia and normalizes levels of p-Akt, phosphatidylinositol 3,4,5-trisphosphate, and active caspase-3. Neutrophils from in vivo G-CSF–treated G6pc3−/− mice exhibit increased glucose uptake and elevated intracellular levels of G6P, lactate, and adenosine-5′-triphosphate, leading to improved functionality. Together, the results strongly suggest that G-CSF improves G6pc3−/− neutrophil survival by modulating apoptotic mediators and rectifies function by enhancing energy homeostasis.

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.

G6PC3 deficiency, characterized by neutropenia and neutrophil dysfunction, is caused by deficiencies in the endoplasmic reticulum (ER) enzyme glucose-6-phosphatase-β (G6Pase-β or G6PC3) that converts glucose-6-phosphate (G6P) into glucose, the primary energy source of neutrophils. Enhanced neutrophil ER stress and apoptosis underlie neutropenia in G6PC3 deficiency, but the exact functional role of G6Pase-β in neutrophils remains unknown. We hypothesized that the ER recycles G6Pase-β–generated glucose to the cytoplasm, thus regulating the amount of available cytoplasmic glucose/G6P in neutrophils. Accordingly, a G6Pase-β deficiency would impair glycolysis and hexose monophosphate shunt activities leading to reductions in lactate production, adenosine-5′-triphosphate (ATP) production, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Using annexin V–depleted neutrophils, we show that glucose transporter-1 translocation is impaired in neutrophils from G6pc3−/− mice and G6PC3-deficient patients along with impaired glucose uptake in G6pc3−/− neutrophils. Moreover, levels of G6P, lactate, and ATP are markedly lower in murine and human G6PC3-deficient neutrophils, compared with their respective controls. In parallel, the expression of NADPH oxidase subunits and membrane translocation of p47phox are down-regulated in murine and human G6PC3-deficient neutrophils. The results establish that in nonapoptotic neutrophils, G6Pase-β is essential for normal energy homeostasis. A G6Pase-β deficiency prevents recycling of ER glucose to the cytoplasm, leading to neutrophil dysfunction.

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.

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.