ABSTRACT
Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.
Subject(s)
Aspergillus fumigatus/metabolism , Glycerol Kinase/metabolism , Glycerolphosphate Dehydrogenase/metabolism , Oxidative Stress/physiology , Aspergillus fumigatus/genetics , Glucose/metabolism , Glycerol/metabolism , Glycerol Kinase/physiology , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerophosphates , Hydrogen Peroxide/metabolism , Metabolic Networks and Pathways , Mitochondria/metabolism , Oxidation-Reduction , Phenotype , Phosphates/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Metformin is an oral drug widely used for the treatment of type 2 diabetes mellitus. Numerous studies have demonstrated the value of metformin in cancer treatment. However, for metformin to elicit effects on cancer often requires a high dosage, and any underlying mechanism for how to improve its inhibitory effects remains unknown. Here, we found that low mRNA expression of glycerol-3-phosphate dehydrogenase 1 (GPD1) may predict a poor response to metformin treatment in 15 cancer cell lines. In vitro and in vivo, metformin treatment alone significantly suppressed cancer cell proliferation, a phenotype enhanced by GPD1 overexpression. Total cellular glycerol-3-phosphate concentration was significantly increased by the combination of GPD1 overexpression and metformin treatment, which suppressed cancer growth via inhibition of mitochondrial function. Eventually, increased reactive oxygen species and mitochondrial structural damage was observed in GPD1-overexpressing cell lines treated with metformin, which may contribute to cell death. In summary, this study demonstrates that GPD1 overexpression enhances the anticancer activity of metformin and that patients with increased GPD1 expression in tumor cells may respond better to metformin therapy. SIGNIFICANCE: GPD1 overexpression enhances the anticancer effect of metformin through synergistic inhibition of mitochondrial function, thereby providing new insight into metformin-mediated cancer therapy.
Subject(s)
Glycerolphosphate Dehydrogenase/metabolism , Glycerophosphates/metabolism , Metformin/pharmacology , Neoplasms/drug therapy , Neoplasms/metabolism , A549 Cells , Adenosine Triphosphate/biosynthesis , Animals , Antineoplastic Agents/pharmacology , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Respiration/physiology , Drug Synergism , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerolphosphate Dehydrogenase/genetics , HCT116 Cells , Heterografts , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Mitochondria/metabolism , Neoplasms/genetics , Neoplasms/pathology , PC-3 Cells , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Glycerol-3-phosphate (G3P) is the important precursors for triacylglycerol synthesis, while glycerol-3-phosphate dehydrogenase (GPDH) determines the formation of G3P. In this study, two GDPH genes, Dtgdp1 and Dtgdp2 were isolated and identified from Dunaliella tertiolecta. The full-length Dtgdp1 and Dtgdp2 CDS were 2016 bp and 2094 bp, which encoded two putative protein sequences of 671 and 697 amino acids with predicted molecular weights of 73.64 kDa and 76.73 kDa, respectively. DtGDP1 and DtGDP2 both had a close relationship with those of algal and higher plants. DtGDP1 shared two conserved superfamily (A1 and A2) and four signature motifs (I-IV), and the DtGDP2 showed six signature domains (from motif I to VI) and DAO_C conserved family. Our previous work showed that the triethylamine intervention could greatly increase the triacylglycerol content (up to 80%) of D. tertiolecta. This study aims to investigate the effect of triethylamine on GPDH expression. Results showed that, when treated by triethylamine at 100 ppm and 150 ppm, the expression levels of Dtgdp1 and Dtgpd2 were increased to 5.121- and 56.964-fold compared with the control, respectively. Triethylamine seemed to enhance lipid metabolic flow by inducing the expressions of Dtgdp1 and Dtgdp2 to increase the lipid content, which provides a new insight into the desired pathway of lipid synthesis in algae through genetic engineering.
Subject(s)
Chlorophyceae/drug effects , Chlorophyceae/metabolism , Ethylamines/metabolism , Gene Expression Regulation, Plant/drug effects , Glycerolphosphate Dehydrogenase/biosynthesis , Lipid Metabolism/drug effects , Amino Acid Motifs , Chlorophyceae/enzymology , Chlorophyceae/genetics , Glycerolphosphate Dehydrogenase/chemistry , Glycerolphosphate Dehydrogenase/genetics , Molecular WeightABSTRACT
Chloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule.
Subject(s)
Antimalarials/pharmacology , Chloroquine/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Active Transport, Cell Nucleus , Cell Line , Drug Resistance, Fungal/genetics , Glutathione/chemistry , Glycerolphosphate Dehydrogenase/biosynthesis , HEK293 Cells , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/genetics , Phosphorylation/drug effects , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction/drug effects , Stress, Physiological/drug effects , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Transcription Factors/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
α-Tocopheryl succinate (TOS), a redox-silent analogue of vitamin E, suppresses cell growth in a number of clinical and experimental cancers, inhibits mitochondrial succinate dehydrogenase (SDH) and activates reactive oxygen species (ROS) generation. The aim of this study was to test whether TOS also inhibits glycerol-3-phosphate dehydrogenase (mGPDH), another flavoprotein-dependent enzyme of the mitochondrial respiratory chain because there are differences between electron transfer pathway from SDH and mGPDH to coenzyme Q pool. For our experiments brown adipose tissue mitochondria with high expression of mGPDH were used. Our data showed that inhibition of glycerol-3-phosphate (GP)-dependent oxygen consumption by TOS was more pronounced than the succinate (SUC)-dependent one (50% inhibition was reached at 10 µmol/l TOS vs. 80 µmol/l TOS, respectively). A comparison of the inhibitory effect of TOS on GP-oxidase, GP-cytochrome c oxidoreductase and GP-dehydrogenase activities showed that TOS directly interacts with the dehydrogenase. After TOS application the GP-dependent generation of ROS was highly depressed. It may thus be concluded that TOS-induced inhibition of mGPDH is more pronounced than TOS-induced inhibition of SDH and that the inhibitory effect of TOS for both substrates is exerted at different locations of the particular dehydrogenases. Our data indicate that the inhibition of mGPDH activity could also play a role in TOS-induced growth suppression in neoplastic cells.
Subject(s)
Carcinogenesis/genetics , Glycerolphosphate Dehydrogenase/biosynthesis , Mitochondria/enzymology , alpha-Tocopherol/administration & dosage , Adipose Tissue, Brown/enzymology , Animals , Cricetinae , Gene Expression Regulation, Neoplastic/drug effects , Glycerolphosphate Dehydrogenase/antagonists & inhibitors , Humans , Mitochondria/drug effects , Neoplasms/drug therapy , Neoplasms/enzymology , Oxygen Consumption/drug effects , Reactive Oxygen Species/metabolismABSTRACT
The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-ß, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-ß, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of ß-actin, glycerophosphate dehydrogenase, and ß2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.
Subject(s)
Interferon Inducers/pharmacology , Interferons/biosynthesis , RNA, Double-Stranded/pharmacology , RNA, Fungal/pharmacology , Transcription, Genetic/drug effects , Transcriptional Activation/drug effects , 2',5'-Oligoadenylate Synthetase/biosynthesis , 2',5'-Oligoadenylate Synthetase/genetics , Actins/biosynthesis , Actins/genetics , Adult , Antiviral Agents/pharmacology , Cell Line , Child , Fibroblasts/metabolism , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerolphosphate Dehydrogenase/genetics , Gossypol/analogs & derivatives , Gossypol/pharmacology , Humans , Interferon alpha-2 , Interferon-alpha/pharmacology , Interferon-beta/biosynthesis , Interferon-beta/genetics , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interferons/genetics , Lymphocytes/metabolism , Maus Elberfeld virus/drug effects , Recombinant Proteins/pharmacology , beta 2-Microglobulin/biosynthesis , beta 2-Microglobulin/genetics , eIF-2 Kinase/biosynthesis , eIF-2 Kinase/geneticsABSTRACT
We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24 h decreased by 40 % the early differentiation marker CCAAT/enhancer-binding protein ß. However, upon longer-term (10 day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose-response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor γ, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10 day effect of cinacalcet exposure for the first 6, 24, 48 h, 6, and 10 days. Our observations suggest that regardless of the period of exposure, 10 day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established.
Subject(s)
Adipocytes/metabolism , Adipogenesis/drug effects , Naphthalenes/pharmacology , Receptors, Calcium-Sensing/metabolism , Adipogenesis/physiology , Adiponectin/biosynthesis , Adiponectin/metabolism , Adipose Tissue/cytology , Adipose Tissue/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Line, Tumor , Cinacalcet , Cytokines/metabolism , Enzyme Activation , Fatty Acid Synthases/biosynthesis , Fatty Acid Synthases/metabolism , Female , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerolphosphate Dehydrogenase/metabolism , Humans , Inflammation , Male , PPAR gamma/biosynthesis , PPAR gamma/metabolismABSTRACT
We investigated whether lactic acid bacteria isolated from gajami sik-hae (GLAB) are capable of reducing the intracellular lipid accumulation by downregulating the expression of adipogenesis-related genes in differentiated 3T3-L1 cells. The GLAB, Lactobacillus plantarum LG42, significantly decreased the intracellular triglyceride storage and the glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent manner. mRNA expression of transcription factors like peroxisome proliferator-activated receptor (PPAR) γ and CCAAT/enhancer-binding protein (C/EBP) α involved in adipogenesis was markedly decreased by the GLAB treatment. Moreover, the GLAB also decreased the expression level of adipogenic markers like adipocyte fatty acid binding protein (aP2), leptin, GPDH, and fatty acid translocase (CD36) significantly. These results suggest that the GLAB inhibits lipid accumulation in the differentiated adipocyte through downregulating the expression of adipogenic transcription factors and other specific genes involved in lipid metabolism.
Subject(s)
Adipocytes/drug effects , Adipogenesis/drug effects , Lactobacillus plantarum/chemistry , Lipid Metabolism/drug effects , 3T3-L1 Cells , Adipocytes/metabolism , Animals , Down-Regulation/drug effects , Gene Expression Regulation, Developmental/drug effects , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerolphosphate Dehydrogenase/genetics , Lactobacillus plantarum/metabolism , Mice , PPAR gamma/biosynthesis , PPAR gamma/genetics , Transcription Factor AP-2/biosynthesis , Transcription Factor AP-2/geneticsABSTRACT
Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Carrier Proteins/metabolism , Osmosis/drug effects , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , Signal Transduction/drug effects , Transcription, Genetic/drug effects , Animals , Caenorhabditis elegans Proteins/genetics , Carrier Proteins/genetics , Eukaryotic Initiation Factor-2/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Gene Silencing , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerolphosphate Dehydrogenase/genetics , Hypertonic Solutions , Phosphorylation , Protein Kinases/genetics , Signal Transduction/physiology , WNK Lysine-Deficient Protein Kinase 1ABSTRACT
Streptococcus pneumoniae is the most common cause of severe bacterial meningitis in children, the elderly, and immunocompromised individuals. To identify virulence factors preferentially expressed during meningitis, we conducted niche-specific genome-wide in vivo transcriptomic analysis after intranasal infection of mice with serotype 4 or 6A pneumococci. The expression of 34 bacterial genes was substantially altered in brain tissue of mice infected with either of the 2 strains. Ten upregulated genes were common to both strains, 7 of which were evaluated for their role in the development of meningitis. One previously uncharacterized protein, α-glycerophosphate oxidase (GlpO), was cytotoxic for human brain microvascular endothelial cells (HBMECs) via generation of H(2)O(2). A glpO deletion mutant was defective in adherence to HBMECs in vitro as well as in progression from the blood to the brain in vivo. Mutant bacteria also induced markedly reduced meningeal inflammation and brain pathology compared with wild type, despite similar levels of bacteremia. Immunization of mice with GlpO protected against invasive pneumococcal disease and provided additive protection when formulated with pneumolysin toxoid. Our results provide the basis of a strategy that can be adapted to identify genes that contribute to the development of meningitis caused by other pathogens.
Subject(s)
Antigens, Bacterial/biosynthesis , Glycerolphosphate Dehydrogenase/biosynthesis , Meningitis, Pneumococcal/enzymology , Pneumococcal Vaccines/metabolism , Streptococcus pneumoniae/enzymology , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Bacterial Proteins/pharmacology , Cells, Cultured , Female , Gene Expression Regulation, Bacterial/immunology , Gene Expression Regulation, Enzymologic/immunology , Glycerolphosphate Dehydrogenase/genetics , Glycerolphosphate Dehydrogenase/immunology , Humans , Meningitis, Pneumococcal/genetics , Meningitis, Pneumococcal/immunology , Meningitis, Pneumococcal/prevention & control , Mice , Mutation , Pneumococcal Vaccines/immunology , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/immunology , Streptolysins/immunology , Streptolysins/pharmacology , Toxoids/immunology , Toxoids/pharmacologyABSTRACT
Correlation analysis demonstrated a statistically significant correlation of linear-weight characteristics of the steelhead rainbow trout (cultivated steelhead form) with an RNA/DNA ratio and the expression level of the gene encoding cytochrome c oxidase (CO) in two-year-old individuals (1+) as well as the expression level of the gene encoding the myosin heavy chain (MyHC) and activities of the enzymes CO and lactate dehydrogenase (LDH) in muscles and 1-glycerophosphate dehydrogenase (1-GPDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the liver of two- and three-year-old individuals (1+ and 2+). With age, the correlation of 1-GPDH and G-6-PDH activities in the rainbow trout liver with the fish body weight increased, whereas their correlations with the body length reduced. The age- and sex-related distinctions in the MyHC gene expression and activities of the white muscle enzyme LDH and the liver enzymes 2-GPDH and G-6-PDH were detected in rainbow trout of both age cohorts.
Subject(s)
Aging/metabolism , Oncorhynchus mykiss/growth & development , Animals , Body Weights and Measures , DNA/biosynthesis , Electron Transport Complex IV/biosynthesis , Electron Transport Complex IV/genetics , Female , Fisheries , Glucosephosphate Dehydrogenase/biosynthesis , Glucosephosphate Dehydrogenase/genetics , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerolphosphate Dehydrogenase/genetics , Liver/enzymology , Liver/metabolism , Male , Muscle, Skeletal/enzymology , Muscle, Skeletal/metabolism , Myosin Heavy Chains/biosynthesis , Myosin Heavy Chains/genetics , Oncorhynchus mykiss/metabolism , RNA/biosynthesis , Sex CharacteristicsABSTRACT
KlNDE1 and KlNDI1 code for two inner mitochondrial membrane transdehydrogenases involved in the maintenance of the intracellular NAD(P)H redox balance. The function of these genes during the utilization of fermentative and respiratory carbon sources was studied. During growth in glucose, deletion of KlNDE1 and KlNDI1 led to an altered kinetic of ethanol and glycerol accumulation compared with the wild type; in addition, KlndiDelta was unable to grow in respiratory substrates. Northern analysis and GFP-fusion experiments showed that KlNDE1 and KlNDI1 regulate the expression of KlGUT2, a component of the glycerol-3-phosphate shuttle. Moreover, both genes seem to be involved in the biogenesis of the mitochondrial tubular network.
Subject(s)
Gene Expression Regulation, Fungal , Glycerolphosphate Dehydrogenase/biosynthesis , Kluyveromyces/enzymology , Kluyveromyces/physiology , Membrane Transport Proteins/metabolism , Oxidoreductases/metabolism , Ethanol/metabolism , Gene Deletion , Glycerol/metabolism , Kluyveromyces/growth & development , Kluyveromyces/metabolism , Membrane Transport Proteins/genetics , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Oxidoreductases/geneticsABSTRACT
Glycerol-3-phosphate dehydrogenase (GPDH) isozymes are differentially expressed among tissues and during flight development. GPDH-1 is involved in the flight-muscle metabolism and GPDH-2 provides precursors for lipid biosynthesis in many tissues. We have isolated and characterized from Triatoma infestans, a Chagas disease vector, two cDNAs encoding for GPDH-1 and GPDH-2 isozymes. The inferred amino acid sequences showed high identity with other GPDH sequences from flying insects. A GPDH-2 transcript was found in fifth instar nymphs, thoracic muscles, adult gonads, and fat bodies. Both isozymes are present in 30-day-old adult thoracic muscle transcripts, and the pattern of expression differs between sexes. The expression of GPDH-1 begins earlier in females, and GPDH-2 is expressed more abundantly in female adult thoracic muscles than in those from males. This finding is consistent with those of other investigators who showed a higher flight initiation probability in T. infestans females than in males.
Subject(s)
Flight, Animal/physiology , Glycerolphosphate Dehydrogenase/biosynthesis , Insect Vectors/enzymology , Isoenzymes/biosynthesis , Triatoma/embryology , Triatoma/enzymology , Animals , Chagas Disease/parasitology , Disease Vectors , Gene Expression , Insect Vectors/growth & development , Triatoma/growth & developmentABSTRACT
The ability of catecholamines to maximally stimulate adipocyte lipolysis (lipolytic capacity) is decreased in obesity. It is not known whether the lipolytic capacity is determined by the ability of adipocytes to differentiate. The aim of the study was to investigate if lipolytic capacity is related to preadipocyte differentiation and if the latter can predict lipolysis in mature adipocytes. IN VITRO experiments were performed on differentiating preadipocytes and isolated mature adipocytes from human subcutaneous adipose tissue. In preadipocytes, noradrenaline-induced lipolysis increased significantly until terminal differentiation (day 12). However, changes in the expression of genes involved in lipolysis (hormone sensitive lipase, adipocyte triglyceride lipase, the alpha2-and beta1-adrenoceptors, perilipin, and fatty acid binding protein) reached a plateau much earlier during differentiation (day 8). A significant positive correlation between lipolysis in differentiated preadipocytes and mature adipocytes was observed for noradrenaline (r=0.5, p<0.01). The late differentiation capacity of preadipocytes measured as glycerol-3-phosphate dehydrogenase activity was positively correlated with noradrenaline-induced lipolysis in preadipocytes (r=0.51, p<0.005) and mature fat cells (r=0.35, p<0.05). In conclusion, intrinsic properties related to terminal differentiation determine the ability of catecholamines to maximally stimulate lipolysis in fat cells. The inability to undergo full differentiation might in part explain the low lipolytic capacity of fat cells among the obese.
Subject(s)
Adipocytes/metabolism , Cell Differentiation/physiology , Lipolysis/physiology , Stem Cells/physiology , Adipocytes/drug effects , Adult , Biomarkers , Body Mass Index , Cell Differentiation/drug effects , Culture Media, Serum-Free , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerolphosphate Dehydrogenase/metabolism , Humans , Lipolysis/drug effects , Male , Neurosecretory Systems/cytology , Neurosecretory Systems/physiology , Norepinephrine/pharmacology , PPAR gamma/biosynthesis , PPAR gamma/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Stem Cells/drug effectsABSTRACT
In order to conduct a physiological functional study of lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (GPDH), we engineered a CHO dhfr(-) cell, by overexpressing either the anti-sense LDH-A RNA (anti-LDH cells) or GPDH (GP3 cells), or both (GP3/anti-LDH cells). LDH activity in the cell cytosol, and lactate content and pHe change in the growth media were found to decrease according to the order: cell lines GP3/anti-LDH > anti-LDH > GP3 > CHO. Intracellular ATP contents, representing the extent of respiration rate, also decreased, according to a rank order as follows: GP3 > CHO > GP3/anti-LDH > anti-LDH. We also attempted to identify and characterize any physiological changes occurring in the cells which harbored diverse metabolic pathways. First, anti-LDH cells with heightened respiration rates were found to display a higher degree of sensitivity to the prooxidant tert-butyl hydroperoxide (tBOOH), and the mitochondrial complex III inhibitor, antimycin A, than the GPDH-expressing cells (GP3 and GP3/anti-LDH), which have a lower respiration rate. Second, the anti-sense LDH-A RNA-expressing cells (anti-LDH and GP3/anti-LDH) evidenced a higher degree of resistance to apoptosis by cell-cell contact inhibition, and a faster doubling time ( approximately 19 h compared with approximately 26 h) than the CHO and GP3 cells. Additionally, cell growth in an extended culture under HCO(3) (-)-free conditions to induce a steep acidification could be maintained with the anti-sense LDH-A RNA-expressing cells, but could not be maintained with the CHO and GP3 cells. Third, we observed that the most appropriate cell line for the optical production of a certain therapeutic protein (Tissue-Plasminogen Activator) was the GP3/anti-LDH cells. Collectively, our data indicate a variety of physiological roles for LDH and GPDH, including cellular acidosis, oxidoresistance, apoptosis by both acidosis and cell-cell contact inhibition, cell growth, and the generation of recombinant proteins.
Subject(s)
Glycerolphosphate Dehydrogenase/biosynthesis , L-Lactate Dehydrogenase/antagonists & inhibitors , Animals , Apoptosis , Bicarbonates/metabolism , CHO Cells , Cell Proliferation , Cricetinae , Cricetulus , Glycerolphosphate Dehydrogenase/genetics , Glycolysis , Hydrogen-Ion Concentration , L-Lactate Dehydrogenase/biosynthesis , L-Lactate Dehydrogenase/genetics , Oxidation-Reduction , RNA, Antisense/genetics , RNA, Messenger/genetics , Recombinant Proteins/biosynthesis , Tissue Plasminogen Activator/biosynthesisABSTRACT
Under anaerobic conditions S. cerevisiae produces glycerol to regenerate NAD(+) from the excess NADH produced in cell metabolism. We here report on the role of an uncharacterized protein, Yig1p (Ypl201cp), in anaerobic glycerol production. Yig1p was previously shown to interact in two-hybrid tests with the GPP1 and GPP2 encoded glycerol 3-phosphatase (Gpp), and we here demonstrate that strains overexpressing YIG1 show strongly decreased Gpp activity and content of the major phosphatase, Gpp1p. However, cells overexpressing YIG1 exhibited only slightly decreased GPP1 transcript levels, suggesting that Yig1p modulates expression on both transcriptional and post-transcriptional levels. In agreement with such a role, a GFP-tagged derivate of Yig1p was localized to both the cytosol and the nucleus. Deletion or overexpression of YIG1 did not, however, significantly affect growth yield or glycerol yield in anaerobic batch cultures, which is consistent with the previously proposed low flux control exerted at the Gpp level.
Subject(s)
Glycerol/metabolism , Phosphoric Monoester Hydrolases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Anaerobiosis , Blotting, Northern , Blotting, Western , DNA, Fungal/genetics , DNA, Fungal/metabolism , Gene Expression , Glycerolphosphate Dehydrogenase/biosynthesis , Glycerolphosphate Dehydrogenase/genetics , Glycerolphosphate Dehydrogenase/metabolism , Oxidation-Reduction , Phosphoric Monoester Hydrolases/biosynthesis , Phosphoric Monoester Hydrolases/genetics , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Based on the principle of the pathway engineering, a novel pathway of producing glycerol was built in E. coli. The gpd1 gene encoding glycerol 3-phosphate dehydrogenase and the hor2 gene encoding glycerol 3-phosphatase were cloned from Saccharomyces cerevisiae, respectively. The two genes were inserted into expression vector pSE380 together. A recombinant plasmid pSE-gpd1-hor2 containing polycistron was constructed under the control of the strong trc promoter. Then it was transformed into E. coli BL21. The result showed the recombinant microorganism GxB-gh could convert glucose to glycerol directly. And the recombinant microorganism GxB-gh was incubated to produce glycerol from D-glucose in the fermentor. The maximal concentration of glycerol was 46.67g/L at 26h. Conversion rate of glucose was 42.87%. The study is about "green" producing glycerol by recombinant microorganism and is also useful for further working in recombining microorganism of producing 1,3-propanediol.
Subject(s)
Escherichia coli/metabolism , Fungal Proteins/genetics , Glycerolphosphate Dehydrogenase/genetics , Phosphoric Monoester Hydrolases/genetics , Saccharomyces cerevisiae/genetics , Cloning, Molecular , Escherichia coli/genetics , Fermentation , Fungal Proteins/biosynthesis , Genetic Engineering , Glycerol/metabolism , Glycerolphosphate Dehydrogenase/biosynthesis , Phosphoric Monoester Hydrolases/biosynthesis , Saccharomyces cerevisiae/enzymologyABSTRACT
Thyroid hormones are important regulators of mitochondrial metabolism. Due to their complex mechanism of action, the timescale of different responses varies from minutes to days. In this work, we studied selective T3 induction of the inner mitochondrial membrane enzyme-glycerophosphate dehydrogenase (mGPDH) in liver of euthyroid rats. We correlated the kinetics of the T3 level in blood, the mRNA level in liver, the activity and amount of mGPDH in liver mitochondria after a single dose of T3. The T3 level reached maximum after 1 h (80 nmol/l) and subsequently rapidly decreased. mGPDH mRNA increased also relatively fast, reaching a maximum after 12 h and fell to the control level after 72 h. An increase of mGPDH activity could be already found after 6 h and reached a maximum after 24 h in accordance with the increase in mGPDH content (2.4-fold vs. 2.7-fold induction). After 72 h, the mGPDH activity showed a significant 30% decrease. When the rats received three subsequent doses of T3, the increase of mGPDH activity was 2-fold higher than after a single T3 dose. The results demonstrate that mGPDH displays rapid induction as well as decay upon disappearance of a hormonal stimulus, indicating a rather short half-life of this inner mitochondrial membrane enzyme.
Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , Glycerolphosphate Dehydrogenase/biosynthesis , Mitochondria, Liver/enzymology , Triiodothyronine/administration & dosage , Animals , Enzyme Activation/drug effects , Gene Expression Regulation, Enzymologic/physiology , Male , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Triiodothyronine/bloodABSTRACT
Effects of functionally gradient calcium phosphate consisting of hydroxyapatite (HAP) and alpha-tricalcium phosphate (alpha-TCP) on proliferation and differentiation of osteoblasts were evaluated using MC3T3-E1 cells. There were no significant differences in the proliferation of MC3T3-E1 cells among HAP-alpha-TCP functionally gradient calcium phosphate, pure HAP, and cell culture plastic wells. mRNA expressions of type I collagen, alkaline phosphate, and osteocalcine were evaluated as indexes of initial; mid-stage, and late-stage osteoblastic differentiation. Basically, HAP-alpha-TCP functionally gradient calcium phosphate and pure HAP enhanced the expressions of the three markers when compared with that of cell culture plastic wells. For type I collagen and alkaline phosphate expressions, HAP-alpha-TCP functionally gradient calcium phosphate showed the same expression level as pure HAP. For osteocalcine expression, HAP-alpha-TCP functionally gradient calcium phosphate showed a higher level than pure HAP. We concluded, therefore, HAP-alpha-TCP functionally gradient calcium phosphate has good potential to be a bone filler material with high osteoconductivity.
Subject(s)
Bone Substitutes/pharmacology , Calcium Phosphates/pharmacology , Ceramics/pharmacology , Durapatite/pharmacology , Osteoblasts/drug effects , 3T3 Cells , Alkaline Phosphatase/biosynthesis , Analysis of Variance , Animals , Bone Substitutes/chemistry , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Ceramics/chemistry , Collagen Type I/biosynthesis , Durapatite/chemistry , Glycerolphosphate Dehydrogenase/biosynthesis , Mice , Osteoblasts/metabolism , Osteocalcin/biosynthesis , RNA, Messenger/biosynthesisABSTRACT
The Saccharomyces cerevisiae PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C. Cells deleted for PLC1 ( plc1Delta) are viable, but display several phenotypes, including osmotic, temperature, and nocodazole sensitivity. We have used a two-hybrid screen to identify Plc1p-interacting proteins. One of the interacting proteins found was Sgd1p, a recently identified, essential, nuclear protein. The SGD1 gene was originally cloned by complementation of an osmostress-sensitive mutant. The Plc1p-Sgd1p interaction was confirmed biochemically by affinity chromatography. SGD1 interacts genetically with both PLC1 and HOG1 (which encodes an osmosensing mitogen-activated protein kinase). Overexpression of Sgd1p suppresses the temperature sensitivity of cells bearing the plc1-4 allele, and the double mutant strain plc1Delta sgd1-1 displays enhanced temperature and nocodazole sensitivity. The plc1Delta hog1Delta strain displays increased osmosensitivity, and has a synthetic defect in glycerol synthesis and the expression of GPD1 (which encodes the enzyme glycerol 3-phosphate dehydrogenase that is involved in glycerol biosynthesis), suggesting that Plc1p and Hog1p function in independent pathways. The hog1Delta sgd1-1 double mutant displays enhanced osmosensitivity relative to that of either single mutant. The triple mutant plc1Delta hog1Delta sgd1-1 is inviable, while the plc1Delta hog1Delta sgd1-2 strain grows extremely slowly and is more osmosensitive than the plc1Delta hog1Delta or hog1Delta sgd1-2 strain. These results are consistent with a model in which Plc1p and Hog1p function in parallel pathways affecting osmoregulation, and signals from both these pathways converge, at least partly, on Sgd1p.