Activities Of Key Glycolytic Enzymes Research Articles

The enhancing effects of selenomethionine on harmine in attenuating pathological cardiac hypertrophy via glycolysis metabolism.

Pathological cardiac hypertrophy, a common feature in various cardiovascular diseases, can be more effectively managed through combination therapies using natural compounds. Harmine, a β-carboline alkaloid found in plants, possesses numerous pharmacological functions, including alleviating cardiac hypertrophy. Similarly, Selenomethionine (SE), a primary organic selenium source, has been shown to mitigate cardiac autophagy and alleviate injury. To explores the therapeutic potential of combining Harmine with SE to treat cardiac hypertrophy. The synergistic effects of SE and harmine against cardiac hypertrophy were assessed invitro with angiotensin II (AngII)-induced hypertrophy and invivo using a Myh6R404Q mouse model. Co-administration of SE and harmine significantly reduced hypertrophy-related markers, outperforming monotherapies. Transcriptomic and metabolic profiling revealed substantial alterations in key metabolic and signalling pathways, particularly those involved in energy metabolism. Notably, the combination therapy led to a marked reduction in the activity of key glycolytic enzymes. Importantly, the addition of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) did not further potentiate these effects, suggesting that the antihypertrophic action is predominantly mediated through glycolytic inhibition. These findings highlight the potential of SE and harmine as a promising combination therapy for the treatment of cardiac hypertrophy.

Potential Effects of NO-Induced Hypoxia-Inducible Factor-1α on Yak Meat Tenderness during Post-Mortem Aging.

The objective of this study was to investigate the mechanism underlying nitric oxide (NO)-induced hypoxia-inducible factor-1α (HIF-1α) and its impact on yak muscle tenderness during post-mortem aging. The Longissimus thoracis et lumborum (LTL) muscle of yak were incubated at 4 °C for 0, 3, 6, 9, 12, 24, and 72 h after treatment with 0.9% saline, NO activator, or a combination of the NO activator and an HIF-1α inhibitor. Results indicated that elevated NO levels could increase HIF-1α transcription to achieve stable expression of HIF-1α protein (P < 0.05). Additionally, elevated NO triggered HIF-1α S-nitrosylation, which further upregulated the activity of key glycolytic enzymes, increased glycogen consumption, accelerated lactic acid accumulation, and decreased pH (P < 0.05). These processes eventually improved the tenderness of yak muscle during post-mortem aging (P < 0.05). The results demonstrated that NO-induced activation of HIF-1α S-nitrosylation enhanced glycolysis during post-mortem aging and provided a possible pathway for improving meat tenderness.

Energy metabolic mechanisms for high altitude sickness: Downregulation of glycolysis and upregulation of the lactic acid/amino acid-pyruvate-TCA pathways and fatty acid oxidation

Hypobaric hypoxia is often associated with the plateau environment and can lead to altitude sickness or death. The underlying cause is a lack of oxygen, which limits energy metabolism and leads to a compensatory stress response. Although glycolysis is commonly accepted as the primary energy source during clinical hypoxia, our preliminary experiments suggest that hypobaric hypoxia may depress glycolysis. To provide a more comprehensive understanding of energy metabolism under short-term hypobaric hypoxia, we exposed mice to a simulated altitude of 5000 m for 6 or 12 h. After the exposure, we collected blood and liver tissues to quantify the substrates, enzymes, and metabolites involved in glycolysis, lactic acid metabolism, the tricarboxylic acid cycle (TCA), and fatty acid β-oxidation. We also performed transcriptome and enzymatic activity analyses of the liver. Our results show that 6 h of hypoxic exposure significantly increased blood glucose, decreased lactic acid and triglyceride concentrations, and altered liver enzyme activities of mice exposed to hypoxia. The key enzymes in the glycolytic, TCA, and fatty acid β-oxidation pathways were primarily affected. Specifically, the activities of key glycolytic enzymes, such as glucokinase, decreased significantly, while the activities of enzymes in the TCA cycle, such as isocitrate dehydrogenase, increased significantly. Lactate dehydrogenase, pyruvate carboxylase, and alanine aminotransferase were upregulated. These changes were partially restored when the exposure time was extended to 12 h, except for further downregulation of phosphofructokinase and glucokinase. This study demonstrates that acute high altitude hypoxia upregulated the lactic acid/amino acid-pyruvate-TCA pathways and fatty acid oxidation, but downregulated glycolysis in the liver of mice. The results obtained in this study provide a theoretical framework for understanding the mechanisms underlying the pathogenesis of high-altitude sickness in humans. Additionally, these findings have potential implications for the development of prevention and treatment strategies for altitude sickness.

Alpha-ketoglutarate partially alleviates effects of high-fat high-fructose diet in mouse muscle.

Consumption of high-calorie diets leads to excessive accumulation of storage lipids in adipose tissue. Metabolic changes occur not only in adipose tissue but in other tissues, too, such as liver, heart, muscle, and brain. This study aimed to explore the effects of high-fat high-fructose diet (HFFD) alone and in the combination with alpha-ketoglutarate (AKG), a well-known cellular metabolite, on energy metabolism in the skeletal muscle of C57BL/6J mice. Five-month-old male mice were divided into four groups - the control one fed a standard diet (10 % kcal fat), HFFD group fed a high-fat high-fructose diet (45 % kcal fat, 15 % kcal fructose), AKG group fed a standard diet with 1 % sodium AKG in drinking water, and HFFD + AKG group fed HFFD and water with 1 % sodium AKG. The dietary regimens lasted 8 weeks. Mice fed HFFD had higher levels of storage triacylglycerides, lower levels of glycogen, and total water-soluble protein, and higher activities of key glycolytic enzymes, namely hexokinase, phosphofructokinase, and pyruvate kinase, as compared with the control group. The results suggest that muscles of HFFD mice may suffer from lipotoxicity. In HFFD + AKG mice, levels of the metabolites and activities of glycolytic enzymes did not differ from the respective values in the control group, except for the activity of pyruvate kinase, which was significantly lower in HFFD + AKG group compared with the control. Thus, metabolic changes in mouse skeletal muscles, caused by HFFD, were alleviated by AKG, indicating a protective role of AKG regarding lipotoxicity.

Valorisation of the Inhibitory Potential of Fresh and Dried Fruit Extracts of Prunus spinosa L. towards Carbohydrate Hydrolysing Enzymes, Protein Glycation, Multiple Oxidants and Oxidative Stress-Induced Changes in Human Plasma Constituents.

Prunus spinosa fruits (sloes), both fresh and dried, are underexplored dietary components and ethno-phytotherapeutic remedies applied to treat chronic oxidative-stress-related diseases, including diabetes. The present study aimed to evaluate drying-related changes in the antidiabetic potential of sloe extracts and some bioactivity mechanisms, which might be connected with their traditional application. The polyphenol-enriched extracts, prepared by fractionated extraction and phytochemically standardised, i.a., by LC-MS/MS, were tested in vitro using a set of biological and chemical models. The experiments revealed the significant extracts’ ability to counteract the generation of advanced glycation end products (AGEs) and inhibit the activity of key glycolytic enzymes, i.e., α-glucosidase and α-amylase. Moreover, they were proved to effectively scavenge multiple oxidants of physiological importance (O2•−, HO•, H2O2, NO•, HOCl), increase the non-enzymatic antioxidant capacity of human plasma (NEAC) under oxidative stress conditions induced by peroxynitrite, and protect plasma proteins and lipids against peroxidation and nitration at in vivo-relevant levels (1–50 µg/mL, equivalent to 0.03–6.32 µg polyphenols/mL). In most cases, the activity of fresh fruit extracts surpassed that of dried-based products. The correlation studies and tests on model compounds proved polyphenols as dominant contributors to the observed effects. Furthermore, the co-occurring representatives of various polyphenolic classes were found to contribute to the biological activity of sloes through additive and synergistic effects. Considering the extraction yield and activity parameters, especially the superior outcomes compared to anti-diabetic drugs aminoguanidine and acarbose in the anti-glycation and α-glucosidase inhibition tests, the methanol–water (75:25, v/v) extract of fresh fruits and its phenolic-enriched fractions revealed the most advantageous potential for functional application.

Shoutai Wan Improves Embryo Survival by Regulating Aerobic Glycolysis of Trophoblast Cells in a Mouse Model of Recurrent Spontaneous Abortion.

Background During embryo implantation, the blastocyst exhibits a high capacity for aerobic glycolysis, which results in a unique microenvironment of high lactate/low pH at the maternal-fetal interface. Shoutai Wan (STW) is an effective Chinese herbal formula widely used in the clinical treatment of recurrent spontaneous abortion (RSA). However, the specific molecular mechanism by which STW prevents abortion is yet to be elucidated. Methods Female CBA/J mice were allocated into six groups randomly and then mated with BALB/c mice as the control group, DBA/2 mice as the RSA model, CBA/J×DBA/2 mice treated with dydrogesterone as the DQYT group, or CBA/J×DBA/2 mice treated with low, medium, and high-dose STW as the STW-L, STW-M, and STW-H groups, respectively. Drug administration started 14 days before mating and ended on the 14th day of pregnancy. The embryo loss rate of each group was calculated on day 14 of gestation, and the pregnancy outcomes of the mice in each group were observed. The mouse serum was collected to determine the levels of progesterone (P) and chorionic gonadotropin (CG). The activities of HK2, PKM2, and LDHA, the key glycolytic enzymes in each group, were detected. The expressions of lactate, ATP, HK2, PKM2, LDHA, MCT4, GLUT1, and GPR81 as well as the morphology of trophoblast cells were examined. Results The embryo loss rate and adverse pregnancy outcomes were significantly increased (P < 0.05) in the RSA model group. After dydrogesterone or different doses of STW treatment, the embryo loss rate and adverse pregnancy outcomes were rescued to varying degrees (P < 0.05). Interestingly, there was no significant difference among the groups in terms of serum P and CG (P < 0.05). Moreover, the activities of key glycolytic enzymes, lactate, ATP, HK2, PKM2, LDHA, MCT4, GLUT1, GPR81 protein or mRNA expression, and morphological abnormalities of trophoblast cells improved significantly in the RSA mice after dydrogesterone or different doses of STW treatment (P < 0.05). Conclusion STW can promote aerobic glycolysis in trophoblast cells of RSA mouse embryos, thereby improving the microenvironment of the maternal-fetal interface and enhancing embryo implantation.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, II-III, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

Middle aged turn point in parameters of oxidative stress and glucose catabolism in mouse cerebellum during lifespan: minor effects of every-other-day fasting.

The cerebellum is considered to develop aging markers more slowly than other parts of the brain. Intensification of free radical processes and compromised bioenergetics, critical hallmarks of normal brain aging, may be slowed down by caloric restriction. This study aimed to evaluate the intensity of oxidative stress and the enzymatic potential to utilize glucose via glycolysis or the pentose phosphate pathway (PPP) in the cerebellum of mice under ad libitum versus every-other-day fasting (EODF) feeding regimens. Levels of lipid peroxides, activities of antioxidant and key glycolytic and PPP enzymes were measured in young (6-month), middle-aged (12-month) and old (18-month) C57BL/6J mice. The cerebellum showed the most dramatic increase in lipid peroxide levels, antioxidant capacity and PPP key enzyme activities and the sharpest decline in the activities of key glycolytic enzymes under transition from young to middle age but these changes slowed when transiting from middle to old age. A decrease in the activity of the key glycolytic enzyme phosphofructokinase was accompanied by a concomitant increase in the activities of hexokinase and glucose-6-phosphate dehydrogenase (G6PDH), which may suggest that during normal cerebellar aging glucose metabolism shifts from glycolysis to the pentose phosphate pathway. The data indicate that intensification of free radical processes in the cerebellum occurred by middle age and that activation of the PPP together with increased antioxidant capacity can help to resist these changes into old age. However, the EODF regime did not significantly modulate or alleviate any of the metabolic processes studied in this analysis of the aging cerebellum.

Middle age as a turning point in mouse cerebral cortex energy and redox metabolism: Modulation by every-other-day fasting.

Normal brain aging is accompanied by intensification of free radical processes and compromised bioenergetics. Caloric restriction is expected to counteract these changes but the underlying protective mechanisms remain poorly understood. The present work aimed to investigate the intensity of oxidative stress and energy metabolism in the cerebral cortex comparing mice of different ages as well as comparing mice given one of two regimens of food availability: ad libitum versus every-other-day fasting (EODF). Levels of oxidative stress markers, ketone bodies, glycolytic intermediates, mitochondrial respiration, and activities of antioxidant and glycolytic enzymes were assessed in cortex from 6-, 12- and 18-month old C57BL/6J mice. The greatest increase in oxidative stress markers and the sharpest decline in key glycolytic enzyme activities was observed in mice upon the transition from young (6months) to middle (12months) age, with smaller changes occurring upon transition to old-age (18months). Brain mitochondrial respiration showed no significant changes with age. A decrease in the activities of key glycolytic enzymes was accompanied by an increase in the activity of glucose-6-phosphate dehydrogenase suggesting that during normal brain aging glucose metabolism is altered to lower glycolytic activity and increase dependence on the pentose-phosphate pathway. Interestingly, levels of ketone bodies and antioxidant capacity showed a greater decrease in the brain cortex of females as compared with males. The EODF regimen further suppressed glycolytic enzyme activities in the cortex of old mice, and partially enhanced oxygen consumption and respiratory control in the cortex of middle aged and old males. Thus, in the mammalian cortex the major aging-induced metabolic changes are already seen in middle age and are slightly alleviated by an intermittent fasting mode of feeding.

Effects of prolonged cold-ischemia on autophagy in the graft lung in a rat orthotopic lung transplantation model

IntroductionIschemia-reperfusion (I/R) injury causes present challenges in the field of graft transplantation which is also a major contributor to early graft dysfunction or failure after organ transplantation. The study focuses on the effects of prolonged cold-ischemia (CI) on the autophagic activity in the graft lung in a rat orthotopic lung transplantation model. Material and methodsDonor lungs were preserved under CI conditions for different periods. An orthotopic lung transplantation model was developed, and the lung tissues from donor lungs subjected to CI preservation and reperfusion were harvested. We evaluated the effects of different CI periods on autophagy, reactive oxygen species (ROS) and glucose consumption. Additionally, the mechanism by which prolonged CI affected autophagy was investigated through determination of the molecules related to the mTOR pathway after treatment with 3-Methyladenine (3-MA), rapamycin and an adenosine triphosphate (ATP) synthase inhibitor oligomycin (OM). ResultsProlonged CI led to increased activities of key glycolytic enzymes, glucose consumption and lactic acid production. Autophagy, ROS and glucose consumption were induced in the graft lung after I/R, which reached peak levels after 6 h and was gradually decreased. Most importantly, the perfusion treatment of 3-MA or OM decreased ROS level and autophagy, but increased the extent of mTOR phosphorylation, while the perfusion treatment of rapamycin induced ROS and autophagy. ConclusionTaken together, autophagy mediated by a prolonged CI preservation affects the glucose consumption and ROS production in the graft lung via the mTOR signaling pathway.

Every-Other-Day Feeding Decreases Glycolytic and Mitochondrial Energy-Producing Potentials in the Brain and Liver of Young Mice.

Intermittent fasting is used to reduce body mass in obese adult humans and animals. However, information on the impact of one type of intermittent fasting (IF) called every-other-day feeding (EODF) on young animals is scarce. In this study, 1-month-old mice of both sexes were subjected to a 4-week regimen of EODF using age-matched counterparts fed ad libitum as controls. At the end of EODF exposure, experimental male and female mice weighed 14 and 13% less than the control counterparts. The EODF regimen resulted in lower liver levels of glycogen, glucose, and lactate, but did not affect lactate level in mouse cerebral cortex of both sexes. Activities of key glycolytic enzymes (hexokinase, phosphofructokinase, and pyruvate kinase) in liver of experimental mice were lower than those in controls. In the cerebral cortex, only hexokinase and pyruvate kinase activities were lower than in controls, but phosphofructokinase activity was not affected in IF females and was higher in IF males as compared with ad libitum fed males. Mitochondria isolated from liver of IF mice had lower respiratory control ratios, but those from the cortex had the same values as control animals. The concentration of β-hydroxybutyrate and the activity of β-hydroxybutyrate dehydrogenase were lower in the IF mouse liver, but not changed or enhanced in the IF cerebral cortex. Thus, animal responses to IF do not depend significantly on sex and are directed to decrease energy metabolism to save resources, and the effects are more pronounced in the liver than in the brain.

Effects of acute hyperglycaemia stress on indices of glucose metabolism and glucose transporter genes expression in cobia ( Rachycentron canadum )

The objective of the present study is to preliminarily clarify the mechanism of carbohydrates metabolism in cobia (Rachycentron canadum) (85 ± 3 g) receiving injection of glucose solution. We examined plasma glucose (GLU), total protein (TP), triglyceride (TG), cholesterol (CHOL), insulin, liver glycogen and muscle glycogen, activities of hepatic hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), as well as relative expressions of glucose transporter 1 (GLUT1), GLUT2, GLUT3, GLUT4, GLUT5 and GLUT9 in hemocyte, liver and muscle of R. canadum when fish were injected with 200 μl of glucose solution (255 mg/ml) after 0, 1, 2, 4, 8, 12, 24 and 48 hr. Fish received injection of 0.68% saline served as control. Results indicated that the plasma GLU, TG and CHOL increased and reached peak at 1, 8 and 48 hr postinjection (hpi) respectively. The hepatic glycogen increased from 1 hpi, and reached peak at 8 hpi, plasma insulin increased at 1 hpi, and reached peak at 2 hpi, and activity of hepatic PK peaked at 8 hpi. Furthermore, the relative expressions of GLUT1, GLUT2, GLUT3, GLUT4 and GLUT5 in hemocytes reached peak at 1,4, 8, 4 and 8 hpi, respectively, relative expressions of GLUT2, GLUT3, GLUT5 and GLUT9 in liver reached peak at 24, 24, 12 and 24 hpi, respectively, and relative expressions of GLUT1 and GLUT3 in muscle were significantly higher at 2 and 2–4 hpi, respectively compared with those in controls. In conclusion, low ability of utilizing glucose in R. canadum may be attributed to insufficient insulin secretion, low activities of key glycolytic enzymes (HK, PFK and PK) regulated by glucose injection and slow increase of GLUTs.

Aberrant mitochondrial bioenergetics in the cerebral cortex of the Fmr1 knockout mouse model of fragile X syndrome.

Impaired energy metabolism may play a role in the pathogenesis of neurodevelopmental disorders including fragile X syndrome (FXS). We checked brain energy status and some aspects of cell bioenergetics, namely the activity of key glycolytic enzymes, glycerol-3-phosphate shuttle and mitochondrial respiratory chain (MRC) complexes, in the cerebral cortex of the Fmr1 knockout (KO) mouse model of FXS. We found that, despite a hyperactivation of MRC complexes, adenosine triphosphate (ATP) production via mitochondrial oxidative phosphorylation (OXPHOS) is compromised, resulting in brain energy impairment in juvenile and late-adult Fmr1 KO mice. Thus, an altered mitochondrial energy metabolism may contribute to neurological impairment in FXS.

Prolonged Cold-Ischemia Increases Reactive Oxygen Species and Activates Autophagy by Enhancing Glycolysis in the Graft through the mTOR Signaling Pathway in Orthotopic Lung Transplantation of Rats

Purpose Ischemia-reperfusion (I/R) injury continues to plague the field of graft transplantation and is a major contributor to early graft dysfunction or failure after organ transplantation. The aim of the present study is to investigate the mechanisms by which prolonged CI influences the autophagy in the graft lung, in which the mTOR signaling pathway was involved. Methods Orthotopic lung transplantation models were induced in rats, whose lungs were preserved under CI conditions for varying durations. Autophagy activities, energy metabolism, and reactive oxygen species (ROS) level were determined at each time point. The perfusion treatments of inhibitor (3-Methyladenine [3-MA]) and activator (rapamycin) of autophagy and inhibitor of oxidative phosphorylation (oligomycin [OM]) were performed to verify the relationships among mTOR signaling pathway and autophagy in the cold I/R injury. Results Autophagy was induced by CI preservation, which was peaked at 12 h after CI. Additionally, a coexistence of reduced oxidative phosphorylation and elevated ROS level was observed in rat lung tissues. Increased activities of key glycolytic enzymes, glucose consumption and lactic acid production followed with prolonged CI, which became stable after a 9 h period of CI. Of importance, the perfusion treatment of 3-MA or OM decreased ROS level and Beclin-1, PINK1, Parkin, and AMPK expression, but increased mTOR expression; while the perfusion treatment of rapamycin induced a reverse tendency. Conclusion These findings demonstrate that activated autophagy during prolonged CI preservation leads to poor oxidative phosphorylation, robust glycolysis, and ROS level rise in the graft lung via the mTOR signaling pathway.

Global Profiling of Lysine Acetylation in Borrelia burgdorferi B31 Reveals Its Role in Central Metabolism.

The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ΔackA (acetyl-P- and acetyl-CoA-) and B31-A3 Δpta (acetyl-P+ and acetyl-CoA-) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ΔackA mutant, 140 proteins were acetylated in the Δpta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.

Modulation of GLO1 Expression Affects Malignant Properties of Cells.

The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

Key glycolytic enzyme activities of skeletal muscle are decreased under fed and fasted states in mice with knocked down levels of Shc proteins.

Shc proteins interact with the insulin receptor, indicating a role in regulating glycolysis. To investigate this idea, the activities of key glycolytic regulatory enzymes and metabolites levels were measured in skeletal muscle from mice with low levels of Shc proteins (ShcKO) and wild-type (WT) controls. The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase were decreased in ShcKO versus WT mice under both fed and fasted conditions. Increased alanine transaminase and branched-chain amino acid transaminase activities were also observed in ShcKO mice under both fed and fasting conditions. Protein expression of glycolytic enzymes was unchanged in the ShcKO and WT mice, indicating that decreased activities were not due to changes in their transcription. Changes in metabolite levels were consistent with the observed changes in enzyme activities. In particular, the levels of fructose-2,6-bisphosphate, a potent activator of phosphofructokinase-1, were consistently decreased in the ShcKO mice. Furthermore, the levels of lactate (inhibitor of hexokinase and phosphofructokinase-1) and citrate (inhibitor of phosphofructokinase-1 and pyruvate kinase) were increased in fed and fasted ShcKO versus WT mice. Pyruvate dehydrogenase activity was lower in ShcKO versus WT mice under fed conditions, and showed inhibition under fasting conditions in both ShcKO and WT mice, with ShcKO mice showing less inhibition than the WT mice. Pyruvate dehydrogenase kinase 4 levels were unchanged under fed conditions but were lower in the ShcKO mice under fasting conditions. These studies indicate that decreased levels of Shc proteins in skeletal muscle lead to a decreased glycolytic capacity in both fed and fasted states.

Short-term periodic feed deprivation in Labeo rohita fingerlings: Effect on the activities of digestive, metabolic and anti-oxidative enzymes

This study aimed to determine the effect of periodic feed deprivation on the activities of digestive, metabolic and anti-oxidative enzymes of Labeo rohita (commonly known as rohu) fingerlings. Fish (average weight, 3.73±0.06g) were periodically starved for 0 (0day-per-week, dpw), 1 (1dpw), 2 (2dpw) or 3 (3dpw) consecutive day (s) per week for a total duration of 10weeks. On the day of feeding, fish were fed to satiation twice daily with a diet containing 30% crude protein and 6% lipid. Results showed increased protease and amylase activities in the 1dpw and 2dpw groups, suggesting that feeding fish for 6 or 5days a week markedly improved the digestive capacity of fish when compared to daily feeding (0dpw). However, the activities of lipase and alkaline phosphatase did not respond significantly (P>0.05) due to periodic feed deprivation. Periodic feed deprivation of 1dpw did not lead to any changes in the metabolic and anti-oxidative enzyme activities of the fish. In fingerlings deprived of feed for 2dpw, higher hepatic glycolytic enzyme activities, hexokinase and pyruvate kinase, were observed but with no change in the activities of gluconeogenic (glucose-6-phosphatase, aspartate amino-transferase and alanine amino-transferase) and Kreb's cycle (malate dehydrogenase) enzymes. The group deprived of feed for 3dpw exhibited lower activities of malate dehydrogenase, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase. Increased activities of anti-oxidative enzymes, superoxide dismutase and catalase, in the 2 and 3dpw groups in this study implied an adaptive response of L. rohita fingerlings to cope with the oxidative stress caused due to feed deprivation. Higher activities of protease and amylase in the 1 and 2dpw groups, and higher key glycolytic enzyme activities in the 2dpw group would suggest improved digestive and metabolic functions, however, feed deprivation beyond 2dpw seemed to have adverse effect on L. rohita fingerlings.

Activities of key glycolytic enzymes in the plasma of Saudi autistic patients

Activities of key glycolytic enzymes in the plasma of Saudi autistic patients A El-Ansary1, S Al-Daihan1, A Al-Dabas1, L Al-Ayadhi21Biochemistry Department, Science College, 2Autism Research and Treatment Unit, Department of Physiology, Faculty of Medicine, King Saud University, Riyadh, Saudi ArabiaObjective: Measurement of plasma levels of lactate, lactate oxidase (LOX), pyruvate kinase (PK), and hexokinase (HK) as possible glycolytic parameters to assess brain damage in autistic patients.Design and methods: Plasmatic levels of lactate, LOX, PK, and HK were determined in 20 autistic children aged 3&ndash;15 years and 20 age-matching healthy control subjects.Results: Plasmatic levels of lactate and LOX were significantly higher in autistic patients compared to healthy subjects and that of PK and HK were significantly lower in these patients as compared to controls. This could reflect the impaired metabolism of astrocytes, the brain cells responsible for the production and provision of lactate, as the primary metabolic fuel for neurons.Conclusion: Remarkably different levels of plasma glycolytic parameters were recorded in Saudi autistic patients. This could be correlated to the impairment of energy metabolism, glutathione depletion, and lead intoxication previously detected in the same investigated samples. The identification of biochemical markers related to autism would be advantageous for earlier clinical diagnosis and intervention.Keywords: autism, glycolysis, lactate, lactate oxidase, pyruvate kinase, hexokinase

Effect of capsaicin on glucose metabolism studied in experimental lung carcinogenesis

In the present study, we have assessed the chemopreventive effect of capsaicin (CAP) on glucose metabolism with reference to blood glucose and liver glycogen levels, key glycolytic, and gluconeogenic enzymes along with electron transport chain (ETC) complexes during benzo(a)pyrene (B(a)P)-induced lung cancer in Swiss albino mice. B(a)P (50 mg kg−1 body weight)-induced lung cancer animals showed marked decline in blood glucose levels, glycogen levels, elevations in the activities of key glycolytic enzymes (hexokinase, phosphoglucoisomerase and aldolase), and gluconeogenic enzymes (glucose-6-phosphatase and fructose-6-phosphatase) together with a decrease in the activities of ETC complexes. Supplementation of CAP (10 mg kg−1 body weight) inhibited all the above alterations during lung cancer and restored near normalcy. Histochemical analysis by periodic acid Schiff's staining further confirmed the biochemical findings that highlighted the chemopreventive action of CAP during B(a)P-induced experimental lung tumourigenesis.