Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Metabolic regulation through the endosomal system

The endosomal system plays an essential role in cell homeostasis by controlling cellular signaling, nutrient sensing, cell polarity and cell migration. However, its place in the regulation of tissue, organ and whole body physiology is less well understood. Recent studies have revealed an important role for the endosomal system in regulating glucose and lipid homeostasis, with implications for metabolic disorders such as type 2 diabetes, hypercholesterolemia and non‐alcoholic fatty liver disease. By taking insights from in vitro studies of endocytosis and exploring their effects on metabolism, we can begin to connect the fields of endosomal transport and metabolic homeostasis. In this review, we explore current understanding of how the endosomal system influences the systemic regulation of glucose and lipid metabolism in mice and humans. We highlight exciting new insights that help translate findings from single cells to a wider physiological level and open up new directions for endosomal research.     

The synthesis of N‐benzoylindoles as inhibitors of rat erythrocyte glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase

Glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) play an important function in various biochemical processes as they generate reducing power of the cell. Thus, metabolic reprogramming of reduced nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis is reported to be a vital step in cancer progression as well as in combinational therapeutic approaches. In this study, N‐benzoylindoles 9a‐ ‐ 9d , which form the main framework of many natural indole derivatives such as indomethacin and N‐benzoylindoylbarbituric acid, were synthesized through three easy and effective steps as an in vitro inhibitor effect of G6PD and 6PGD. The N‐benzoylindoles inhibited the enzymatic activity with IC₅₀ in the range of 3.391505 μM for G6PD and 2.19–990 μM for 6PGD.     

Induction of apoptosis by Drosophila Myc

Myc proteins are essential regulators of cellular growth and proliferation during normal development. Activating mutations in myc genes result in excessive growth and are frequently associated with human cancers. At the same time, forced expression of Myc sensitizes vertebrate cells towards different pro-apoptotic stimuli. Recently, the ability of overexpressed Myc to induce cell-autonomous apoptosis has been shown to be evolutionarily conserved in Drosophila Myc (dMyc). Here, we show that dMyc induced apoptosis is accompanied by the induction of Drosophila p53 mRNA, but that dp53 activity is not essential for dMyc's ability to induce apoptosis. Conversely, larvae carrying a hypomorphic dmyc mutation are more resistant to the apoptosis-promoting effects of X-irradiation. These data suggest that the control of apoptosis is a physiological function of Myc and that dMyc might play a role in the response to DNA damage.     

Effect of dexamethasone on neutrophil metabolism

The effect of dexamethasone on glucose and glutamine metabolism was investigated. The consumption and oxidation of glucose and glutamine, and the production of glutamate and lactate were determined in neutrophils cultured for 3 h in the presence of dexamethasone. The activities and expression of glucose-6-phosphate dehydrogenase (G6PDH) and phosphate-dependent glutaminase were also determined under the same conditions. Addition of dexamethasone to the culture medium caused a significant increase of glucose consumption at 0.5 microm (123.9%) and 1.0 microm (78.3%) concentrations. In spite of this, however, glucose oxidation remained unchanged. The glucocorticoid did not change glutamine consumption but caused a significant increase of glutamate production and did not alter glutamine oxidation. Dexamethasone-treated neutrophils had a significant decrease of G6PDH activity and expression in particular at 1.0 microm concentration. Phosphate- dependent glutaminase activity was also decreased (about 34%) by dexamethasone treatment. A similar effect was observed on glutaminase expression as indicated by RT-PCR analysis. Thus, the effect of dexamethasone on neutrophil metabolism was particularly noticeable with respect to G6PDH and glutaminase activities where a decrease in the respective mRNA levels was demonstrated.     

Purification of glucose‐6‐phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme on which the pentose phosphate pathway was checked. In this study, purification of a G6PD enzyme was carried out by using rat erythrocytes with a specific activity of 13.7 EU/mg and a yield of 67.7 and 155.6‐fold by using 2′,5′‐ADP Sepharose‐4B affinity column chromatography. For the purpose of identifying the purity of enzyme and molecular mass of the subunit, a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was carried out. The molecular mass of subunit was calculated 56.5 kDa approximately. Then, an investigation was carried out regarding the inhibitory effects caused by various metal ions (Fe²⁺, Pb²⁺, Cd²⁺, Ag⁺, and Zn²⁺) on G6PD enzyme activities, as per Beutler method at 340 nm under in vitro conditions. Lineweaver–Burk diagrams were used for estimation of the IC₅₀ and K_i values for the metals. K_i values for Pb⁺², Cd⁺², Ag⁺, and Zn⁺² were 113.3, 215.2, 19.4, and 474.7 μM, respectively.     

Glucose‐6‐phosphate dehydrogenase is posttranslationally regulated in the larvae of the freeze‐tolerant gall fly,Eurosta solidaginis,in response to freezing

The freeze‐tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) undergo substantial alterations to their molecular physiology during the winter including the production of elevated quantities of glycerol and sorbitol, which function as cryoprotectants to survive whole body freezing. Production of these cryoprotectants depends on cytosolic pools of nicotinamide adenine dinucleotide phosphate H (NADPH), a major source being the pentose phosphate pathway (PPP). Glucose‐6‐phosphate dehydrogenase (G6PDH) mediates the rate‐limiting and committed step of the PPP and therefore its molecular properties were explored in larvae sampled from control versus frozen states. G6PDH was purified from control (5°C) and frozen (?15°C) E. solidaginis larvae by a single‐step chromatography method utilizing 2′,5′‐ADP agarose and analyzed to determine its enzymatic parameters. Studies revealed a decrease in K_m for G6P in the frozen animals (to 50% of control values) suggesting an increased flux through the PPP. Immunoblotting of the purified enzyme showed differences in the relative extent of several posttranslational modifications, notably ubiquitination (95% decrease in frozen larvae), cysteine nitrosylation (61% decrease), threonine (4.1 fold increase), and serine phosphorylation (59% decrease). Together these data suggested that the increased flux through the PPP needed to generate NADPH for cryoprotectants synthesis is regulated, at least in part, through posttranslational alterations of G6PDH.     

Effect of astaxanthin and aluminum chloride on erythrocyte G6PD and 6PGD enzyme activities in vivo and on erythrocyte G6PD in vitro in rats

In this study, we investigated the effect of astaxanthin (Ast) and aluminum (Al) on the erythrocyte glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activities in vivo and on G6PD enzyme in vitro in rats. For in vitro studies, G6PD enzyme was purified from rat erythrocyte by using 2′,5′‐ADP‐Sepharose 4B affinity gel. The effects of Ast and Al³⁺ ion were investigated on the purified enzyme. It was determined that Ast increased the enzyme activity, whereas Al³⁺ inhibited the enzyme activity noncompetitively (IC₅₀ values; 0.679 mM, K_i values 1.32 mM). For in vivo studies, the rats were divided into the groups: control (Cont.), Al, Ast, and Al + Ast. The last three groups were compared with the control group. In Al group, a significant degree of inhibition was observed in the activity of G6PD and 6PGD enzymes when compared with the control group (P < 0.05), whereas there was an increase in the activities of G6PD and 6PGD enzymes in Ast and Al + Ast groups (P < 0.05).     

Oxidative damage and antioxidant enzyme activities in experimental hypothyroidism

Free radicals are now well known to damage cellular components. To investigate whether age and thyroid level affect peroxidation speed, we examined the levels of malondialdehyde and antioxidant enzyme activities in different age groups of hypothyroid rats. Hypothyroidism was induced in 30- and 60-day-old Wistar Albino rats by the i.p. administration of propylthiouracil (10 mg kg(-1) body weight) for 15 days. While malondialdehyde levels of 30- or 60-day-old hypothyroid rats were increased in liver, they were decreased in the tissues of the heart and thyroid. While glucose-6-phosphate dehydrogenase activity levels did not change in heart, brain and liver tissues of 30-day-old rats, they increased in brain and heart tissues of 60-day-old experimental groups, but decreased in the liver. Catalase activities decreased in the liver and heart of rats with hypothyroidism, but increased in erythrocytes. In control groups while malondialdehyde levels increased in brain, heart and thymus with regard to age, they decreased in plasma. Glucose-6-phosphate dehydrogenase and catalase activities were not affected by age in tissues of the thymus, thyroid and brain, but they were decreased in the heart tissue. The changes in the levels of lipid peroxidation and antioxidant enzyme activities which were determined in different tissues of hypothyroid rats indicate a cause for functional disorder of these tissues. Moreover, there may be changes depending on age at lipid peroxidation and antioxidant enzyme activity levels.     

Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis

In green tissues of plants under illumination, photosynthesis is the primary source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is utilized in reductive reactions such as carbon fixation and nitrogen assimilation. In non-photosynthetic tissues or under non-photosynthetic conditions, the oxidative pentose phosphate pathway contributes to basic metabolism as one of the major sources of NADPH. The first and committed reaction is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). We characterized the six members of the G6PDH gene family in Arabidopsis. Transit peptide analysis predicted two cytosolic and four plastidic isoforms. Five of the six genes encode active G6PDHs. The recombinant isoforms showed differences in substrate requirements and sensitivities to feedback inhibition. Plastidic isoforms were redox sensitive. One cytosolic isoform was insensitive to redox changes, while the other was inactivated by oxidation. The respective genes had distinct expression patterns that did not correlate with the activity of the proteins, implying a regulatory mechanism beyond the control of mRNA abundance. Two cytosolic and one plastidic isoform were detected in vivo using zymograms, and the respective genes were identified using T-DNA insertion lines. The activity of a plastidic isoform was detected in all tissues including photosynthetic tissues despite its sensitivity to reduction observed in vitro. Genomic data, gene expression, and in vivo enzyme activity data were integrated with in vitro biochemical data to propose in vivo roles for individual G6PDH isoforms in Arabidopsis.     

Possible involvement of NADPH requirement in regulation of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase levels in rat liver

Summary Previous studies examining regulation of synthesis of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase in rat liver have focussed on the induction of these enzymes by different diets and some hormones. However, the precise mechanism regulating increases in the activities of these enzymes is unknown and the factors involved remain unidentified. Considering that many of these metabolic conditions occur simultaneously with the increase of some NADPH consuming pathway, in particular fatty acid synthesis, we suggest that the activities of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase could be regulated through a mechanism involving changes in the NADPH requirement. Here, we have studied the effect of changes in the flux through different NADPH consuming pathways on the NADPH/NADP ratio and on Glucose-6-Phosphate and 6-Phosphogluconate levels. The results show that: i) an increase in consumption of NADPH, caused by activation of fatty acid synthesis or the detoxification system which consumes NADPH, is paralleled by an increase in levels of these enzymes; ii) when increase in consumption of NADPH is prevented, Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - ME Malic Enzyme - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide - t-BHP t-Butyl hydroperoxide - BCNU 1,3,-Bis (2-chloroethyl)-1-nitrosourea - GR Glutathione Dehydrogenase - 2-ME 2-Mercaptoethanol - DTT Dithiothreitol - NADP B-Nicotinamide-Adenine Dinucleotide Phosphate - NADPH B-Nicotinamide-Adenine Dinucleotide Phosphate Reduced - EDTA Ethylenediaminetetraacetic Acid - GSH Glutathione Reduced Form - GSSG Glutathione Oxidized Form     

Drosophila Bcl-2 proteins participate in stress-induced apoptosis, but are not required for normal development

Many developing tissues require programmed cell death (PCD) for proper formation. In mice and C. elegans, developmental PCD is regulated by the Bcl-2 family of proteins. Two bcl-2 genes are encoded in the Drosophila genome (debcl/dBorg1/Drob-1/dBok and buffy/dBorg2) and previous RNAi-based studies suggested a requirement for these in embryonic development. However, we report here that, despite the fact that many tissues in fruit flies are shaped by PCD, deletion of the bcl-2 genes does not perturb normal development. We investigated whether the fly bcl-2 genes regulate non-apoptotic processes that require caspases, but found these to be bcl-2 gene-independent. However, irradiation of the mutants demonstrates that DNA damage-induced apoptosis, mediated by Reaper, is blocked by buffy and that debcl is required to inhibit buffy. Our results demonstrate that developmental PCD regulation in the fly does not rely upon the Bcl-2 proteins, but that they provide an added layer of protection in the apoptotic response to stress.     

In vivo radiolabeling and turnover of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster: Gene dosage effects

In vivo radiolabeling of Drosophila melanogaster sn-glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8; GPDH) has been accomplished by microinjection of ³H-leucine into anesthetized flies. Comigration of immunoprecipitated radiolabeled GPDH with purified ¹⁴C-labeled GPDH-1 in SDS polyacrylamide disc gels has established the monospecificity of our immunoprecipitation technique. Short-term uptake experiments have demonstrated that maximum radiolabel incorporation of total TCA precipitable protein and immunoprecipitable GPDH-1 occurs within 4 hours postinjection, with GPDH-1 accounting for approximately 1% of the total radiolabeled TCA precipitable protein. In order to develop the parameters for turnover studies of GPDH in Drosophila, a comparative analysis of the rates of synthesis and degradation of GPDH-1 in flies bearing two and three doses of the structural gene have been conducted by the construction of adult flies aneuploid and euploid for the cytogenetic region 25F-26B on the left arm of chromosome II. Short-term uptake studies have demonstrated that the rate of GPDH-1 synthesis in the three-dose flies is approximately 1.58 times that found in the two-dose euploid flies. This value is in close agreement with data obtained for steady-state levels of CRM by rocket immunoelectrophoresis. In contrast, longterm pulse-chase experiments have revealed that rates of GPDH-1 degradation in these aneumploid and euploid flies appear to be identical. These data suggest that the rate of GPDH-1 synthesis in Drosophila is primarily regulated by a tightly linked cis-acting element which appears to act autonomously with respect to gene copy number as well as steady-state GPDH protein levels.     

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase‐2

The apoptotic initiator caspase‐2 has been implicated in oocyte death, in DNA damage‐ and heat shock‐induced death, and in mitotic catastrophe. We show here that the mitosis‐promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase‐2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase‐2 interdomain, prevents caspase‐2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase‐2 detected during interphase was lost in mitosis. Expression of S340A non‐phosphorylatable caspase‐2 abrogated mitotic suppression of caspase‐2 and apoptosis in various settings, including oocytes induced to undergo cdk1‐dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase‐2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase‐2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase‐2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur.     

Metabolic regulation of mesenchymal stem cell in expansion and therapeutic application

Human mesenchymal or stromal cells (hMSCs) isolated from various adult tissues are primary candidates in cell therapy and tissue regeneration. Despite promising results in preclinical studies, robust therapeutic responses to MSC treatment have not been reproducibly demonstrated in clinical trials. In the translation of MSC‐based therapy to clinical application, studies of MSC metabolism have significant implication in optimizing bioprocessing conditions to obtain therapeutically competent hMSC population for clinical application. In addition, understanding the contribution of metabolic cues in directing hMSC fate also provides avenues to potentiate their therapeutic effects by modulating their metabolic properties. This review focuses on MSC metabolism and discusses their unique metabolic features in the context of common metabolic properties shared by stem cells. Recent advances in the fundamental understanding of MSC metabolic characteristics in relation to their in vivo origin and metabolic regulation during proliferation, lineage‐specific differentiation, and exposure to in vivo ischemic conditions are summarized. Metabolic strategies in directing MSC fate to enhance their therapeutic potential in tissue engineering and regenerative medicine are discussed. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:468–481, 2015     

Correlated evolution of the cis-acting regulatory elements and developmental expression of the Drosophila Gld gene in seven species from the subgroup melanogaster

The tissue-specific expression patterns of glucose dehydrogenase (GLD) exhibit a high degree of inter specific variation in the adult reproductive tract among the species in the genus Drosophila. We chose to focus on the evolution of GLD expression and the evolution of the Gld promoter in seven closely related species in the mela-nogaster subgroup as a means of elucidating the relationship of changes in cis-acting regulatory elements in the Gld promoter region with changes in tissue-specific expression. Although little variation in tissue-specific patterns of GLD was found in nonreproductive tissues during development, a surprisingly high level of variation was observed in the expression of GLD in both developing and ma-ture reproductive organs. In some cases this variation is correlated with changes in sequence elements in the Gld promoter which were previously shown to direct tissue-specific expression in the reproductive tract. In particular D. teissieri adult males do not express GLD in their ejaculatory ducts, atypical of the melanogaster subgroup species. The Gld promoter region of D. teissieri specifically lacks all three of the TTAGA regulatory elements present in D. melanogaster. The TTAGA elements were previously shown to direct reporter gene expression to the ejaculatory duct. Together these data suggest the absence or presence of the TTAGA elements may be responsible for variation in the absence or presence of GLD in the ejaculatory duct among species. © 1994 Wiley-Liss, Inc.     

Developmental variation in effects of the second and third chromosomes on the activities of the glucose 6-phosphate and 6-phosphogluconate dehydrogenases in Drosophila melanogaster

Developmental profiles of the second- and third-chromosome modifiers of the activities of glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) in Drosophila melanogaster were investigated. Third-chromosome modifiers showed very strong effects on both enzyme activities at larval, pupal, and adult stages, whereas second-chromosome effects were detected mainly at larval and adult stages. For both enzyme activities and both chromosomes, the correlation over line means between larval and pupal stages was significantly positive, but the correlation between larval or pupal stage and adult stage was not significant. This result suggests that the actions of modifiers on G6PD and 6PGD activities are influenced by the change of developmental stages. Correlation between G6PD and 6PGD activities was positive and highly significant throughout the developmental stages for both sets of chromosomes, although third-chromosome correlations were slightly higher than second-chromosome correlations. The magnitude of the correlation between G6PD and 6PGD activities does not seem to be influenced by the change of development. Diallel crosses for both sets of chromosomes indicate that the action of activity modifiers is mainly additive for both sets of chromosomes, but dominance effects were detected in some cases in adult males. Significant maternal effects were detected for the third chromosome for both enzyme activities until the pupal stage. The change of the activity modifier action after emergence of the imago and the significant correlation between G6PD and 6PGD activities were also detected for diallel progeny.This work was supported by Public Health Service Grant NIH-GM11546.Paper No. 10211 of the journal series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695.