Two hexokinases of the shrimp Penaeus (Litopenaeus) vannamei are differentially expressed during oxygen limited conditions.

The white shrimp Penaeus (Litopenaeus) vannamei is the most cultivated shrimp worldwide. Compared to other shrimp species, it has higher resistance to adverse conditions. During hypoxia, the shrimp reduces oxygen consumption and adjusts energy metabolism via anaerobic glycolysis, among other strategies. Hexokinase (HK) is the first enzyme of glycolysis and a key regulation point. In mammals and other vertebrates, there are several tissue-specific HK isoforms with differences in expression and enzyme activity. In contrast, crustacean HKs have been relatively little studied. We studied the P. vannamei HK isoforms during hypoxia and reoxygenation. We cloned two HK1 sequences named HK1-long (1455 bp) and HK1-short (1302 bp), and one HK2 (1344 bp). In normoxia, total HK1 expression is higher in hepatopancreas, while HK2 is higher in gills. Severe hypoxia (1 mg/L of DO) after 12 h exposure and 1 h of reoxygenation increased HK1 expression in both organs, but HK2 expression changed differentially. In hepatopancreas, HK2 expression increased in 6 and 12 h of hypoxia but diminished to normoxia levels after reoxygenation. In gills, HK2 expression decreased after 12 h of hypoxia. HK activity increased in hepatopancreas after 12 h hypoxia, opposite to gills. These results indicate that shrimp HK isoforms respond to hypoxia and reoxygenation in a tissue-specific manner. Intracellular glucose levels did not change in any case, showing the shrimp ability to maintain glucose homeostasis during hypoxia.

Snf1p/Hxk2p/Mig1p pathway regulates hexose transporters transcript levels, affecting the exponential growth and mitochondrial respiration of Saccharomyces cerevisiae.

The Crabtree effect molecular regulation comprehension could help to improve ethanol production with biotechnological purposes and a better understanding of cancer etiology due to its similarity with the Warburg effect. Snf1p/Hxk2p/Mig1p pathway has been linked with the transcriptional regulation of the hexose transporters and phenotypes associated with the Crabtree effect. Nevertheless, direct evidence linking the genetic control of the hexose transporters with modulation of the Crabtree effect phenotypes by the Snf1p/Hxk2p/Mig1p pathway is still lacking. In this sense, we provide evidence that SNF1 and HXK2 genes deletion affects exponential growth, mitochondrial respiration, and transcript levels of hexose transporters in a glucose-dependent manner. The Vmax of the hexose transporters with the high transcript levels was correlated positively with the exponential growth and negatively with the mitochondrial respiration. HXT2 gene transcript levels were the most affected by the deletion of the SNF1/HXK2/MIG1 pathway. Deleting the orthologous genes SNF1 and HXK2 in Kluyveromyces marxianus (Crabtree negative yeast) has an opposite effect compared to Saccharomyces cerevisiae in growth and mitochondrial respiration. Overall, these results indicate that the SNF1/HXK2/MIG1 pathway regulates transcript levels of the hexose transporters, which shows an association with the exponential growth and mitochondrial respiration in a glucose-dependent manner.

Guard-cell-targeted overexpression of Arabidopsis Hexokinase 1 can improve water use efficiency in field-grown tobacco plants.

Water deficit currently acts as one of the largest limiting factors for agricultural productivity worldwide. Additionally, limitation by water scarcity is projected to continue in the future with the further onset of effects of global climate change. As a result, it is critical to develop or breed for crops that have increased water use efficiency and that are more capable of coping with water scarce conditions. However, increased intrinsic water use efficiency (iWUE) typically brings a trade-off with CO2 assimilation as all gas exchange is mediated by stomata, through which CO2 enters the leaf while water vapor exits. Previously, promising results were shown using guard-cell-targeted overexpression of hexokinase to increase iWUE without incurring a penalty in photosynthetic rates or biomass production. Here, two homozygous transgenic tobacco (Nicotiana tabacum) lines expressing Arabidopsis Hexokinase 1 (AtHXK1) constitutively (35SHXK2 and 35SHXK5) and a line that had guard-cell-targeted overexpression of AtHXK1 (GCHXK2) were evaluated relative to wild type for traits related to photosynthesis and yield. In this study, iWUE was significantly higher in GCHXK2 compared with wild type without negatively impacting CO2 assimilation, although results were dependent upon leaf age and proximity of precipitation event to gas exchange measurement.

Prunus Hexokinase 3 genes alter primary C-metabolism and promote drought and salt stress tolerance in Arabidopsis transgenic plants.

Hexokinases (HXKs) and fructokinases (FRKs) are the only two families of enzymes in plants that have been identified as able to phosphorylate Glucose (Glc) and Fructose (Fru). Glc can only be phosphorylated in plants by HXKs, while Fru can be phosphorylated by either HXKs or FRKs. The various subcellular localizations of HXKs in plants indicate that they are involved in diverse functions, including anther dehiscence and pollen germination, stomatal closure in response to sugar levels, stomatal aperture and reducing transpiration. Its association with modulating programmed cell death, and responses to oxidative stress and pathogen infection (abiotic and biotic stresses) also have been reported. To extend our understanding about the function of HXK-like genes in the response of Prunus rootstocks to abiotic stress, we performed a detailed bioinformatic and functional analysis of hexokinase 3-like genes (HXK3s) from two Prunus rootstock genotypes, 'M.2624' (Prunus cerasifera Ehrh × P. munsoniana W.Wight & Hedrick) and 'M.F12/1' (P. avium L.), which are tolerant and sensitive to hypoxia stress, respectively. A previous large-scale transcriptome sequencing of roots of these rootstocks, showed that this HXK3-like gene that was highly induced in the tolerant genotype under hypoxia conditions. In silico analysis of gene promoters from M.2624 and M.F12/1 genotypes revealed regulatory elements that could explain differential transcriptional profiles of HXK3 genes. Subcellular localization was determinates by both bioinformatic prediction and expression of their protein fused to the green fluorescent protein (GFP) in protoplasts and transgenic plants of Arabidopsis. Both approaches showed that they are expressed in plastids. Metabolomics analysis of Arabidopsis plants ectopically expressing Prunus HXK3 genes revealed that content of several metabolites including phosphorylated sugars (G6P), starch and some metabolites associated with the TCA cycle were affected. These transgenic Arabidopsis plants showed improved tolerance to salt and drought stress under growth chamber conditions. Our results suggest that Prunus HXK3 is a potential candidate for enhancing tolerance to salt and drought stresses in stone fruit trees and other plants.

SNF1 controls the glycolytic flux and mitochondrial respiration.

The switch between mitochondrial respiration and fermentation as the main ATP production pathway through an increase glycolytic flux is known as the Crabtree effect. The elucidation of the molecular mechanism of the Crabtree effect may have important applications in ethanol production and lay the groundwork for the Warburg effect, which is essential in the molecular etiology of cancer. A key piece in this mechanism could be Snf1p, which is a protein that participates in the nutritional response including glucose metabolism. Thus, this work aimed to recognize the role of the SNF1 gene on the glycolytic flux and mitochondrial respiration through the glucose concentration variation to gain insights about its relationship with the Crabtree effect. Herein, we found that SNF1 deletion in Saccharomyces cerevisiae cells grown at 1% glucose, decreased glycolytic flux, increased NAD(P)H concentration, enhanced HXK2 gene transcription, and decreased mitochondrial respiration. Meanwhile, the same deletion increased the mitochondrial respiration of cells grown at 10% glucose. Altogether, these findings indicate that SNF1 is important to respond to glucose concentration variation and is involved in the switch between mitochondrial respiration and fermentation.

Biochemical properties and subcellular localization of six members of the HXK family in maize and its metabolic contribution to embryo germination.

BACKGROUND: Seed germination is a crucial process in the plant life cycle when a dramatic variation of type and sugar content occurs just as the seed is hydrated. The production of hexose 6 phosphate is a key node in different pathways that are required for a successful germination. Hexokinase (HXK) is the only plant enzyme that phosphorylates glucose (Glc), so it is key to fueling several metabolic pathways depending on their substrate specificity, metabolite regulatory responses and subcellular localization. In maize, the HXK family is composed of nine genes, but only six of them (ZmHXK4-9) putatively encode catalytically active enzymes. Here, we cloned and functionally characterized putative catalytic enzymes to analyze their metabolic contribution during germination process. RESULTS: From the six HXKs analyzed here, only ZmHXK9 has minimal hexose phosphorylating activity even though enzymatic function of all isoforms (ZmHXK4-9) was confirmed using a yeast complementation approach. The kinetic parameters of recombinant proteins showed that ZmHXK4-7 have high catalytic efficiency for Glc, fructose (Fru) and mannose (Man), ZmHXK7 has a lower Km for ATP, and together with ZmHXK8 they have lower sensitivity to inhibition by ADP, G6P and N-acetylglucosamine than ZmHXK4-6 and ZmHXK9. Additionally, we demonstrated that ZmHXK4-6 and ZmHXK9 are located in the mitochondria and their location relies on the first 30 amino acids of the N-terminal domain. Otherwise, ZmHXK7-8 are constitutively located in the cytosol. HXK activity was detected in cytosolic and mitochondrial fractions and high Glc and Fru phosphorylating activities were found in imbibed embryos. CONCLUSIONS: Considering the biochemical characteristics, location and the expression of ZmHXK4 at onset of germination, we suggest that it is the main contributor to mitochondrial activity at early germination times, at 24 h other ZmHXKs also contribute to the total activity. While in the cytosol, ZmHXK7 could be responsible for the activity at the onset of germination, although later, ZmHXK8 also contributes to the total HXK activity. Our observations suggest that the HXKs may be redundant proteins with specific roles depending on carbon and ATP availability, metabolic needs, or sensor requirements. Further investigation is necessary to understand their specific or redundant physiological roles.

Hypometabolic strategy and glucose metabolism maintenance of Aedes aegypti egg desiccation.

The mosquito Aedes aegypti is vector of several viruses including yellow fever virus, dengue virus chikungunya virus and Zika virus. One of the major problems involving these diseases transmission is that A. aegypti embryos are resistant to desiccation at the end of embryogenesis, surviving and remaining viable for several months inside the egg. Therefore, a fine metabolism control is essential to support these organisms throughout this period of resistance. The carbohydrate metabolism has been shown to be of great importance during arthropod embryogenesis, changing dramatically in order to promote growth and differentiation and in periods of resistance. This study investigated fundamental aspects of glucose metabolism in three stages of A. aegypti egg development: pre-desiccated, desiccated, and rehydrated. The activities of regulatory enzymes in carbohydrate metabolism such as pyruvate kinase, hexokinase and glucose 6-phosphate dehydrogenase were evaluated. We show that these activities were reduced in A. aegypti desiccated eggs, suggesting a decreased activity of glycolytic and pentose phosphate pathway. In contrast, gluconeogenesis increased in desiccated eggs, which uses protein as substrate to synthesize glucose. Accordingly, protein amount decreased during this stage, while glucose levels increased. Glycogen content, a major carbohydrate reserve in mosquitoes, was evaluated and shown to be lower in desiccated and rehydrated eggs, indicating it was used to supply energy metabolism. We observed a reactivation of carbohydrate catabolism and an increased gluconeogenesis after rehydration, suggesting that controlling glucose metabolism was essential not only to survive the period of desiccation, but also for subsequent larvae hatch. Taken together, these results contribute to a better understanding of metabolism regulation in A. aegypti eggs during desiccation periods. Such regulatory mechanisms enable higher survival rate and consequently promote virus transmission by these important disease vectors, making them interesting subjects in the search for novel control methods.

Transcriptional regulation of the protein kinase a subunits in Saccharomyces cerevisiae during fermentative growth.

Yeast cells can adapt their growth in response to the nutritional environment. Glucose is the favourite carbon source of Saccharomyces cerevisiae, which prefers a fermentative metabolism despite the presence of oxygen. When glucose is consumed, the cell switches to the aerobic metabolism of ethanol, during the so-called diauxic shift. The difference between fermentative and aerobic growth is in part mediated by a regulatory mechanism called glucose repression. During glucose derepression a profound gene transcriptional reprogramming occurs and genes involved in the utilization of alternative carbon sources are expressed. Protein kinase A (PKA) controls different physiological responses following the increment of cAMP as a consequence of a particular stimulus. cAMP-PKA is one of the major pathways involved in the transduction of glucose signalling. In this work the regulation of the promoters of the PKA subunits during respiratory and fermentative metabolism are studied. It is demonstrated that all these promoters are upregulated in the presence of glycerol as carbon source through the Snf1/Cat8 pathway. However, in the presence of glucose as carbon source, the regulation of each PKA promoter subunits is different and only TPK1 is repressed by the complex Hxk2/Mig1 in the presence of active Snf1. Copyright © 2017 John Wiley & Sons, Ltd.

High 18F-FDG uptake in PMAH correlated with normal expression of Glut1, HK1, HK2, and HK3.

BACKGROUND: Primary macronodular adrenal hyperplasia (PMAH) is a rare cause of Cushing's syndrome, characterized by functioning adrenal macronodules and variable cortisol production. Recently, we demonstrated a high 18F-FDG uptake in PMAH, an unexpected finding for a benign disorder. PURPOSE: To investigate whether there is a correlation between 18F-FDG high uptake and the expression levels of the glycolytic pathway components GLUT1, HK1, HK2, and HK3 in PMAH. MATERIAL AND METHODS: We selected 12 patients undergoing surgery for PMAH who had preoperatively undergone 18F-FDG PET/CT. mRNA and protein expression of the selected genes were evaluated in the adrenal nodules from patients who underwent surgery through quantitative RT-PCR and by immunohistochemistry, respectively. RESULTS: SUVmax in PMAH was in the range of 3.3-8.9 and the adrenal size was in the range of 3.5-15 cm. A strong correlation between 18F-FDG uptake and largest adrenal diameter was observed in patients with PMAH. However, no correlation between 18F-FDG uptake and GLUT1, HK1, HK2, HK3 mRNA, and protein expression was observed. CONCLUSION: High 18F-FDG uptake is observed in the majority of PMAH cases. However, 18F-FDG uptake in PMAH is independent of the expression levels of GLUT1, HK1, HK2, and HK3. Further investigation is required to elucidate the molecular mechanisms underlying increased 18F-FDG uptake in PMAH.

Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum.

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and ß-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of ß-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and ß-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.

Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.

Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.

Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

Family-based association study between SLC2A1, HK1, and LEPR polymorphisms with myelomeningocele in Chile.

Obese/diabetic mothers present a higher risk to develop offspring with myelomeningocele (MM), evidence supporting the role of energy homeostasis-related genes in neural tube defects. Using polymerase chain reaction-restriction fragment length polymorphism, we have genotyped SLC2A1, HK1, and LEPR single-nucleotide polymorphisms in 105 Chilean patients with MM and their parents in order to evaluate allele-phenotype associations by means of allele/haplotype transmission test (TDT) and parent-of-origin effects. We detected an undertransmission for the SLC2A1 haplotype T-A (rs710218-rs2229682; P = .040), which was not significant when only lower MM (90% of the cases) was analyzed. In addition, the leptin receptor rs1137100 G allele showed a significant increase in the risk of MM for maternal-derived alleles in the whole sample (2.43-fold; P = .038) and in lower MM (3.20-fold; P = .014). Our results support the role of genes involved in energy homeostasis in the risk of developing MM, thus sustaining the hypothesis of diverse pathways and genetic mechanisms acting in the expression of such birth defect.

Metformin reverses hexokinase and phosphofructokinase downregulation and intracellular distribution in the heart of diabetic mice.

Diabetes mellitus is characterized by hyperglycemia and its associated complications, including cardiomyopathy. Metformin, in addition to lowering blood glucose levels, provides cardioprotection for diabetic subjects. Glycolysis is essential to cardiac metabolism and its reduction may contribute to diabetic cardiomyopathy. Hexokinase (HK) and phosphofructokinase (PFK), rate-limiting enzymes of glycolysis, are downregulated in cardiac muscle from diabetic subjects, playing a central role on the decreased glucose utilization in the heart of diabetic subjects. Thus, the aim of this study was to determine whether metformin modulates heart HK and PFK from diabetic mice. Diabetes was induced by streptozotocin injection on male Swiss mice, which were treated for three consecutive days with 250 mg/kg metformin before evaluating HK and PFK activity, expression, and intracellular distribution on the heart of these subjects. We show that metformin abrogates the downregulation of HK and PFK in the heart of streptozotocin-induced diabetic mice. This effect is not correlated to alteration on the enzymes' transcription and expression. However, the intracellular distribution of both enzymes is altered in diabetic hearts that show increased activity of the soluble fraction when compared to the particulate fraction. Moreover, this pattern is reversed upon the treatment with metformin, which is correlated with the effects of the drug on the enzymes activity. Altogether, our results support evidences that metformin alter the intracellular localization of HK and PFK augmenting glucose utilization by diabetic hearts and, thus, conferring cardiac protection to diabetic subjects.

A low-protein diet during pregnancy alters glucose metabolism and insulin secretion.

In pancreatic islets, glucose metabolism is a key process for insulin secretion, and pregnancy requires an increase in insulin secretion to compensate for the typical insulin resistance at the end of this period. Because a low-protein diet decreases insulin secretion, this type of diet could impair glucose homeostasis, leading to gestational diabetes. In pancreatic islets, we investigated GLUT2, glucokinase and hexokinase expression patterns as well as glucose uptake, utilization and oxidation rates. Adult control non-pregnant (CNP) and control pregnant (CP) rats were fed a normal protein diet (17%), whereas low-protein non-pregnant (LPNP) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) from days 1 to 15 of pregnancy. The insulin secretion in 2.8 mmol l(-1) of glucose was higher in islets from LPP rats than that in islets from CP, CNP and LPNP rats. Maximal insulin release was obtained at 8.3 and 16.7 mmol l(-1) of glucose in LPP and CP groups, respectively. The glucose dose-response curve from LPNP group was shifted to the right in relation to the CNP group. In the CP group, the concentration-response curve to glucose was shifted to the left compared with the CNP group. The LPP groups exhibited an "inverted U-shape" dose-response curve. The alterations in the GLUT2, glucokinase and hexokinase expression patterns neither impaired glucose metabolism nor correlated with glucose islet sensitivity, suggesting that ß-cell sensitivity to glucose requires secondary events other than the observed metabolic/molecular events.

Characterization of non-cytosolic hexokinase activity in white skeletal muscle from goldfish (Carassius auratus L.) and the effect of cold acclimation.

HK (hexokinase) is an enzyme involved in the first step in the glucose metabolism pathway, converting glucose into G6P (glucose 6-phosphate). Owing to the importance of skeletal muscle for fish swimming and acclimation processes, we used goldfish (Carassius auratus L.) white muscle in order to investigate subcellular distribution and kinetics of HK. In this study, we report that HK activity is predominantly localized in the mitochondrial fraction [NC-HK (non-cytosolic HK)] in goldfish white muscle. Studies of the kinetic parameters revealed that the Km (Michaelis-Menten constant) for glucose was 0.41±0.03 mM and that for mannose was 3-fold lower, whereas the affinity for fructose was too low to be measured. The Km for ATP was 0.88±0.05 mM, whereas no activity was observed when either GTP or ITP was used as a phosphate donor. A moderate inhibition (20-40%) was found for ADP and AMP. Similar to mammalian HK, G6P and glucose analogues were able to promote an inhibition of between 85 and 100% of activity. Here, we found that acclimation of goldfish at 5°C promoted a 2.5-fold increase in NC-HK compared with its counterpart acclimated at 25°C. However, cytosolic HK activity was not altered after thermal acclimation. In summary, our results suggest that the goldfish has a constitutive NC-HK that shows some similarities to mammalian HK-II and, curiously, may play a role in the broad metabolic changes required during the cold acclimation process.

Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells.

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4, HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4, HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells.

The hexokinase gene family in the zebrafish: structure, expression, functional and phylogenetic analysis.

Hexokinase-catalyzed glucose phosphorylation is the first and crucial step for glucose utilization. Although there are reported studies on glucose metabolism in commercial species, knowledge on it is almost nil in zebrafish (Danio rerio), an important model organism for biological research. We have searched these fish hexokinase genes by BLAST analysis; determined their expression in liver, muscle, brain and heart; measured their response to fasting and glucose administration; and performed homology sequences studies to glimpse their evolutionary history. We have confirmed by RT-qPCR studies that the six DNA sequences annotated as possible hexokinases in the NCBI GenBank are transcribed. The organ distribution of the HXK genes is similar in zebrafish as in mammals, to which they are distantly related. Of these, DrGLK and DrSHXK1 are expressed in the fish liver, DrHXK1 in brain and heart, and DrHXK2 in muscle. The only gene responsive to glucose was liver DrGLK. Its expression is induced approximately 1 h after glucose intraperitoneal injection, but not after saline solution injection. The comparison of the fish sequences and the corresponding mammalian ones imply that in both taxa the main muscle and brain isoforms are fusion products of the ancestral gene, their amino halves having separated before than their carboxy ones, followed by the fusion event, whereas fish and mammalian glucokinase genes remained unduplicated.

A biochemical and genetic study of Leishmania donovani pyruvate kinase.

Here we present a biochemical and molecular biology study of the enzyme pyruvate kinase (PYK) from the parasitic protozoa Leishmania donovani. The PYK gene was cloned, mutagenised and over expressed and its kinetic parameters determined. Like in other kinetoplastids, L. donovani PYK is allosterically stimulated by the effector fructose 2,6 biphosphate and not by fructose 1,6 biphosphate. When the putative effector binding site of L. donovani PYK was mutagenised, we obtained two mutants with extreme kinetic behavior: Lys453Leu, which retained a sigmoidal kinetics and was little affected by the effector; and His480Gln, which deployed a hyperbolic kinetics that was not changed by the addition of the effector. Molecular Dynamics (MD) studies revealed that the mutations not only altered the effector binding site of L. donovani PYK but also changed the folding of its domain C.