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Hypotension is one of the main characteristics of the systemic inflammation, basically caused by endothelial dysfunction. Studies have shown that the amino acid L-kynurenine (KYN) causes vasodilation in mammals, leading to hypotensive shock. In hypotensive shock, when activated by the KYN, the voltage-gated potassium channel encoded by the family KCNQ (Kv7) gene can cause vasodilation. Fructose-1,6-bisphosphate (FBP) it is being considered in studies an anti-inflammatory, antioxidant, immunomodulator, and a modulator of some ion channels (Ca2+, Na+, and K+). We analyzed the effects of KYN and FBP on mean blood pressure (MBP), systolic and diastolic (DBP) blood pressure, and heart rate variability (HRV) in Wistar rats. Results demonstrated that the administration of KYN significant decreased MBP, DBP, and increased HRV. Importantly, the FBP treatment reversed the KYN effects on MBP, DBP, and HRV. Molecular Docking Simulations suggested that KYN and FBP present a very close estimated free energy of binding and the same position into structure of KCNQ4. Our results did demonstrate that FBP blunted the decrease in BP, provoked by KYN. Results raise new hypotheses for future and studies in the treatment of hypotension resulting from inflammation., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Glucose is widely used in the reconstitution of intravenous medications, which often include antimicrobials. How glucose affects antimicrobial activity has not been comprehensively studied. The present work reports that glucose added to bacteria growing in a rich medium suppresses the bactericidal but not the bacteriostatic activity of several antimicrobial classes, thereby revealing a phenomenon called glucose-mediated antimicrobial tolerance. Glucose, at concentrations corresponding to blood-sugar levels of humans, increased survival of Escherichia coli treated with quinolones, aminoglycosides, and cephalosporins with little effect on minimal inhibitory concentration. Glucose suppressed a ROS surge stimulated by ciprofloxacin. Genes involved in phosphorylated fructose metabolism contributed to glucose-mediated tolerance, since a pfkA deficiency, which blocks the formation of fructose-1,6-bisphosphate, eliminated protection by glucose. Disrupting the pentose phosphate pathway or the TCA cycle failed to alter glucose-mediated tolerance, consistent with an upstream involvement of phosphorylated fructose. Exogenous sodium pyruvate or sodium citrate reversed glucose-mediated antimicrobial tolerance. Both metabolites bypass the effects of fructose-1,6-bisphosphate, a compound known to scavenge hydroxyl radical and chelate iron, activities that suppress ROS accumulation. Treatment with these two compounds constitutes a novel way to mitigate the glucose-mediated antimicrobial tolerance that may exist during intravenous antimicrobial therapy, especially for diabetes patients.

To evaluate the effects of fructose diphosphate (FDP) on routine coagulation tests in vitro, we added FDP into the mixed normal plasma to obtain the final concentration of 0, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30 and 35 mg/mL of drug. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FBG) and thrombin time (TT) of samples were analyzed with blood coagulation analyzers from four different manufacturers(Sysmex, Stago, SEKISUI and Werfen) and their corresponding reagents, respectively. Before the experiment, we also observed whether there were significant differences in coagulation test results of different lots of reagents produced by each manufacturer. At the same time as the four routine clotting tests, the Sysmex blood coagulation analyzer and its proprietary analysis software were used to detect the change of maximum platelet aggregation rate in platelet-rich plasma after adding FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL). The results of PT, aPTT and TT showed a FDP (0-35 mg/mL) concentration-dependent increase and a FBG concentration-dependent decrease. The degree of change (increase or decrease) varied depending on the assay system, with PT and aPTT being more affected by the Sysmex blood coagulation testing instrument reagent system and less affected by CEKISUI, TT less affected by CEKISUI and more affected by Stago, and FBG less affected by Stago and more affected by Sysmex. The results of PT, aPTT and TT were statistically positively correlated with their FDP concentrations, while FBG was negatively correlated. The correlation coefficients between FDP and the coagulation testing systems of Sysmex, Stago, Werfen and SEKISUI were 0.975, 0.988, 0.967, 0.986 for PT, and 0.993, 0.989, 0.990 and 0.962 for aPTT, 0.994, 0.960, 0.977 and 0.982 for TT, - 0.990, - 0.983, - 0.989 and - 0.954 for FBG, respectively. Different concentrations of FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL) had different effects on the maximum aggregation rate of platelet induced by the agonists of adenosine diphosphate (ADP, 5 µmol/L), arachidonic acid (Ara, 1 mmol/L), collagen (Col, 2.5 µg/mL) and epinephrine (Epi,10 µmol/L), but the overall downward trend was consistent, that is, with the increase of FDP concentration, the platelet aggregation rate decreased significantly. Our experimental study demonstrated a possible effect of FDP on the assays of coagulation and Platelet aggregation, which may arise because the drug interferes with the coagulation and platelet aggregation detection system, or it may affect our in vivo coagulation system and Platelet aggregation function, the real mechanism of which remains to be further verified and studied., (© 2022. The Author(s).)

Fructose-1,6-bisphosphate (F1,6BP), an intermediate of the glycolytic pathway, has been found to play a promising anticancer effect; nevertheless, the mechanisms involved remain poorly understood. The present study aimed to evaluate the effect and mechanisms of F1,6BP in a human endometrial cancer cell line (Ishikawa). F1,6BP showed an antiproliferative and non-cytotoxic effect on endometrial cancer cells. These effects are related to the increase in reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). These harmful stimuli trigger the upregulation of the expression of pro-apoptotic genes (p53 and Bax), leading to the reduction of cell proliferation through inducing programmed cell death by apoptosis. Furthermore, F1,6BP-treated cells had the formation of autophagosomes induced, as well as a decrease in their proliferative capacity after withdrawing the treatment. Our results demonstrate that F1,6BP acts as an anticancer agent through the generation of mitochondrial instability, loss of cell function, and p53-dependent cell death. Thus, F1,6BP proves to be a potential molecule for use in the treatment against endometrial cancer., (© 2020 John Wiley & Sons, Ltd.)

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

This study aims to investigate the mechanisms through which fructose diphosphate (FDP) causes anti-hypoxia and anti-fatigue effects and improves learning and memory. Mice were divided into three groups: low-dose FDP (FDP-L), high-dose FDP (FDP-H), and a control group. Acute toxic hypoxia induced by carbon monoxide, sodium nitrite, and potassium cyanide and acute cerebral ischemic hypoxia were used to investigate the anti-hypoxia ability of FDP. The tests of rod-rotating, mouse tail suspension, and swimming endurance were used to explore the anti-fatigue effects of FDP. The Morris water maze experiment was used to determine the impact of FDP on learning and memory ability. Poisoning-induced hypoxic tests showed that mouse survival time was significantly prolonged in the FDP-L and FDP-H groups compared with the control group ( p < 0.05). In the exhaustive swimming test, FDP significantly shortened struggling time and prolonged the time of mass-loaded swimming; the rod-rotating test showed that endurance time was significantly prolonged by using FDP ( p < 0.05). FDP significantly decreased lactate and urea nitrogen levels and increased hepatic and muscle glycogen and glucose transporter-4 and Na + -K + -ATPase ( p < 0.05). To conclude, FDP enhances hypoxia tolerance and fatigue resistance and improves learning and memory ability through regulating glucose and energy metabolism.

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta-elemene (β-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated that β-elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. β-Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that β-elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of β-elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose-1,6-bisphosphate (FBP) and L-cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β-elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti-metastatic agent from natural compounds., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicineand John Wiley & Sons Ltd.)

Fat grafting procedures are considered to be a promising regenerative, cell-directed therapy; however, their survival is mainly influenced by ischemia condition. Fructose 1,6-bisphosphate (FBP), as an intermediate in energy metabolism, has the potential to rescue cells and tissues from hypoxic-ischemic circumstances. In the present study, human lipoaspirates were grafted subcutaneously into nude mice followed by a daily intraperitoneal injection of FBP at different doses for 7 days. Next, the grafts were harvested at different time points till 12 weeks postimplantation and were evaluated for cell viability and function, tissue revascularization and inflammatory cell infiltration using histological analysis, whole-mount living tissue imaging, glycerol 3-phosphate dehydrogenase activity assays, and quantitative analysis of gene expression. The results demonstrated that exogenous FBP administration could attenuate the volume and weight reduction of fat graft; meanwhile, FBP enhanced adipocyte viability and function, increased blood vessel formation, and decreased inflammation. Moreover, in vitro cell experiments showed that FBP could promote adipose-derived stem cell viability and vascular endothelial growth factor (VEGF) mRNA expression in ischemia conditions. Our study indicates that FBP can be used as a protective agent for fat grafting and may be applied in stem cell-based regenerative medicine. Stem Cells Translational Medicine 2019;8:606-616., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Background: Although some glycolytic intermediates have been shown to modulate several cell type formation and activation, the functional role of fructose 1,6-bisphosphate (FBP) on osteoclastogenesis is still unknown., Methods: Osteoclastogenesis was evaluated on bone marrow preosteoclasts cultured with M-CSF - 30 ng/ml, RANKL - 10 ng/ml, and two concentrations of FBP (100 and 300 µM). TRAP-positive stained cells were counted, and osteoclastogenic marker genes expression were evaluated by qPCR. Osteoclasts resorption capacity was evaluated by the expression of specific enzymes and capacity to resorb a mineralized matrix. The NF-κB activation was detected using RAW 264.7, stably expressing luciferase on the NF-κB responsive promoter., Results: We show that FBP, the product of the first stage of glycolysis, inhibited RANKL-induced osteoclasts differentiation and TRAP activity. The treatment of preosteoclasts with FBP attenuated osteoclast fusion and formation, without affecting cell viability. Moreover, the inhibition of several osteoclastogenic marker genes expression (TRAP, OSCAR, DC-STAMP, Integrin αv, NFATc1) by FBP correlates with a reduction of mineralized matrix resorption capacity. The mechanism underlying FBP-inhibition of osteoclastogenesis involves NF-κB/NFATc1 signaling pathway inhibition., Conclusion: Altogether these data show a protective role of a natural glycolytic intermediate in bone homeostasis that may have therapeutic benefit for osteolytic diseases.

Sepsis is one of the main causes of hospitalization and mortality in Intensive Care Units. One of the first manifestations of sepsis is encephalopathy, reported in up to 70% of patients, being associated with higher mortality and morbidity. The factors that cause sepsis-associated encephalopathy (SAE) are still not well known, and may be multifactorial, as perfusion changes, neuroinflammation, oxidative stress and glycolytic metabolism alterations. Fructose-1,6-bisphosphate (FBP), a metabolite of the glycolytic route, has been reported as neuroprotective agent. The present study used an experimental sepsis model in C57BL/6 mice. We used in vivo brain imaging to evaluate glycolytic metabolism through microPET scans and the radiopharmaceutical 18 F-fluoro-2-deoxy-D-glucose ( 18 F-FDG). Brain images were obtained before and 12 h after the induction of sepsis in animals with and without FBP treatment. We also evaluated the treatment effects in the brain oxidative stress by measuring the production of reactive oxygen species (ROS), the activity of catalase (CAT) and glutathione peroxidase (GPx), and the levels of fluorescent marker 2'7'-dichlorofluorescein diacetate (DCF). There was a significant decrease in brain glucose metabolism due to experimental sepsis. A significant protective effect of FBP treatment was observed in the cerebral metabolic outcomes. FBP also modulated the production of ROS, evidenced by reduced CAT activity and lower levels of DCF. Our results suggest that FBP may be a possible candidate in the treatment of SAE., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Pulmonary fibrosis is a specific form of interstitial pneumonia. In addition to the idiopathic cause, it may be caused by drugs such as bleomycin (BLM)-used in the treatment of tumors. Fructose-1,6-bisphosphate (FBP) is a high-energy endogenous glycolytic compound that has antifibrotic, anti-inflammatory, and immunomodulatory effects. The aim of this study was to investigate the effects of FBP on both BLM-induced pulmonary fibrosis in mice and in a human embryonic lung fibroblast (MRC-5) culture system. C57BL/6 mice were divided into four groups: control, FBP, BLM, and BLM plus FBP. A single dose of bleomycin (7.5 U/kg) was administered intratracheally, and survival, body weight, Ashcroft score, and histological analysis were evaluated. Pulmonary function and bronchoalveolar lavage fluid (BALF) were also evaluated after a single dose of bleomycin (1.2 U/kg-intratracheally). Treatment with FBP (500 mg/kg) was given on day 0 intraperitoneally. Fibroblasts (MRC-5 cells) were used to access the effect of FBP in vitro. In vivo, FBP increased the survival rate and reduced body weight loss (BLM vs. BLM plus FBP-p < 0.05). FBP also prevented BLM-induced loss of pulmonary function and decreased BALF inflammatory cells, level of fibrosis, and superficial collagen density (p < 0.05). In vitro, FBP (0.62 and 1.25 mM) had inhibitory activity on MRC-5 cells and was able to induce senescence in fibroblasts. These results showed that FBP has the potential of reducing the toxic effects of BLM and may provide supportive therapy for conventional methods used for the treatment of cancer.

Fructose 1, 6-diphosphate (FDP), a glycolytic intermediate，has been identified to possess antioxidant activities. Here we show the protective effect of FDP against alcohol-induced liver injury in mice and the underlying mechanisms. The in vivo experiments demonstrated that FDP, orally administered to mice, dose-dependently suppressed alcohol (50%, v/v, 12ml/kg)-induced increase of serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), serum triglyceride (TG) level and hepatic malondialdehyde (MDA) level. FDP also inhibited liver histological lesions induced by seven-day administration of alcohol to mice. In vitro study indicated that FDP inhibited ethanol-induced L02 cell apoptosis via reducing pro-caspase3 protein level and increasing poly ADP-ribose polymerase (PARP) cleavage. The mechanism analysis showed that FDP prevented ethanol-induced decrease of mouse antioxidant capability through inhibiting the reducion of the level of glutathione (GSH) and activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH-PX) in mouse livers, and suppressing the reducion of GSH level and SOD activity in L02 cells. FDP also enhanced alcohol metabolic rate through increasing alcohol dehydrogenase (ADH) activity and acetaldehyde dehydrogenase (ALDH) protein level, and down-regulating cytochrome p450 2E1 (CYP2E1). These results displayed that FDP protected mice from alcohol-induced liver injury, suggesting the potential activity of FDP in preventing alcoholic liver disease (ALD)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Lactate/lactic acid is an important chemical compound for the manufacturing of bioplastics. The unicellular cyanobacterium Synechocystis sp. PCC 6803 can produce lactate from carbon dioxide and possesses D-lactate dehydrogenase (Ddh). Here, we performed a biochemical analysis of the Ddh from this cyanobacterium (SyDdh) using recombinant proteins. SyDdh was classified into a cyanobacterial clade similar to those from Gram-negative bacteria, although it was distinct from them. SyDdh can use both pyruvate and oxaloacetate as a substrate and is activated by fructose-1,6-bisphosphate and repressed by divalent cations. An amino acid substitution based on multiple sequence alignment data revealed that the glutamine at position 14 and serine at position 234 are important for the allosteric regulation by Mg 2+ and substrate specificity of SyDdh, respectively. These results reveal the characteristic biochemical properties of Ddh in a unicellular cyanobacterium, which are different from those of other bacterial Ddhs.

Many studies of allosteric mechanisms use limited numbers of mutations to test whether residues play "key" roles. However, if a large percentage of the protein contributes to allosteric function, mutating any residue would have a high probability of modifying allostery. Thus, a predicted mechanism that is dependent on only a few residues could erroneously appear to be supported. We used whole-protein alanine-scanning mutagenesis to determine which amino acid sidechains of human liver pyruvate kinase (hL-PYK; approved symbol PKLR) contribute to regulation by fructose-1,6-bisphosphate (Fru-1,6-BP; activator) and alanine (inhibitor). Each nonalanine/nonglycine residue of hL-PYK was mutated to alanine to generate 431 mutant proteins. Allosteric functions in active proteins were quantified by following substrate affinity over a concentration range of effectors. Results show that different residues contribute to the two allosteric functions. Only a small fraction of mutated residues perturbed inhibition by alanine. In contrast, a large percentage of mutated residues influenced activation by Fru-1,6-BP; inhibition by alanine is not simply the reverse of activation by Fru-1,6-BP. Moreover, the results show that Fru-1,6-BP activation would be extremely difficult to elucidate using a limited number of mutations. Additionally, this large mutational data set will be useful to train and test computational algorithms aiming to predict allosteric mechanisms., (© 2017 Wiley Periodicals, Inc.)

Hepatic fibrosis is an extracellular matrix deposition by hepatic stellate cells (HSC). Fibrosis can be caused by iron, which will lead to hydroxyl radical production and cell damage. Fructose-1,6-bisphosphate (FBP) has been shown to deliver therapeutic effects in many pathological situations. In this work, we aimed to test the effects of FBP in HSC cell line, GRX, exposed to an excess of iron (Fe). The Fe-treatment increased cell proliferation and FBP reversed this effect, which was not due to increased necrosis, apoptosis or changes in cell cycle. Oil Red-O staining showed that FBP successfully increased lipid content and lead GRX cells to present characteristics of quiescent HSC. Fe-treatment decreased PPAR-γ expression and increased Col-1 expression. Both effects were reversed by FBP which also decreased TGF-β1 levels in comparison to both control and Fe groups. FBP, also, did not present scavenger activity in the DPPH assay. The treatment with FBP resulted in decreased proliferation rate, Col-1 expression and TGF-β1 release by HSC cells. Furthermore, activated PPAR-γ and increased lipid droplets induce cells to become quiescent, which is a key event to reversion of hepatic fibrosis. FBP also chelates iron showing potential to improve Cell redox state.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver tumor that affects the world population. Liver cancer inevitably causes great harms and its treatment is extremely difficult. Its development is related to the existence of chronic liver injury, such as in cirrhosis. Cancer is a disease related to the process of inflammation so, research with anti-inflammatory agents has been performed for the development of anti-tumor drugs. Fructose-1,6-bisphosphate (FBP), a metabolite of the glycolytic route, has shown anti-inflammatory actions. The purpose of this study is to investigate the effect of FBP on HepG2 cells growth and inflammatory parameters. Results showed that FBP decreased the proliferation of HepG2 cells through trypan blue assay, without causing necrosis, shown by the intracellular release of LDH. By flow cytometry, we observed a significant IL-8 decrease which is closely related to the tumoral progression and chemotherapeutic resistance, especially in HCC. Then, we found, by RT-PCR, a high expression level of pro-apoptotic protein, such as Bax and p53, and decreased the expression levels of anti-apoptotic proteins, like Bcl-2 suggesting apoptosis. Finally, our results showed that FBP can be a potential therapeutic agent to slow the progress of HCC., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The effect of fructose 1,6-bisphosphate (Fru 1,6-P2) on the regulatory enzymes of pentose phosphate pathway of Escherichia coli was examined. Fru 1,6-P2 inhibited E. coli transaldolase (EC 2.2.1.2) competitively against fructose 6-phosphate and uncompetitively against erythrose 4-phosphate, whereas Fru 1,6-P2 did not affect glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Kinetic results can be explained by assuming that transaldolase has two kinds of binding sites for Fru 1,6-P2: a competitive binding site for fructose 6-phosphate and a second binding site on the enzyme-erythrose 4-phosphate complex. Fru 1,6-P2 increased resulting from the stimulation of glycolysis, can inhibit transaldolase and further participates in the elevation of the concentration of ribose 5-phosphate that can be preferentially utilized for anabolic reaction in exponential phase of E. coli., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pyruvate kinase M2 (PKM2) plays a key role in tumor metabolism and regulates the rate-limiting final step of glycolysis. In tumor cells, there are two allosteric effectors for PKM2: fructose-1,6-bisphosphate (FBP) and serine. However, the relationship between FBP and serine for allosteric regulation of PKM2 is unknown. Here we constructed residue/residue fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in bound PKM2 is distinctly different from that in the free state, FBP/PKM2, or Ser/PKM2. The community network analysis indicates that the information can freely transfer from the allosteric sites of FBP and serine to the substrate site in bound PKM2, while there exists a bottleneck for information transfer in the network of the free state. Furthermore, the binding free energy between the substrate and PKM2 for bound PKM2 is significantly lower than either of FBP/PKM2 or Ser/PKM2. Thus, a hypothesis of "synergistic allosteric mechanism" is proposed for the allosteric regulation of FBP and serine. This hypothesis was further confirmed by the perturbational and mutational analyses of community networks and binding free energies. Finally, two possible synergistic allosteric pathways of FBP-K433-T459-R461-A109-V71-R73-MG2-OXL and Ser-I47-C49-R73-MG2-OXL were identified based on the shortest path algorithm and were confirmed by the network perturbation analysis. Interestingly, no similar pathways could be found in the free state. The process targeting on the allosteric pathways can better regulate the glycolysis of PKM2 and significantly inhibit the progression of tumor.

In clinical and experimental settings, a great deal of effort is being made to protect cells and tissues against harmful conditions and to facilitate metabolic recovery following these insults. Much of the recent attention has focused on the protective role of a natural form of sugar, fructose 1,6-bisphosphate (F16bP). F16bP is a high-energy glycolytic intermediate that has been shown to exert a protective action in different cell types and tissues (including the brain, kidney, intestine, liver and heart) against various harmful conditions. For example, there is much evidence that it prevents neuronal damage due to hypoxia and ischemia. Furthermore, the cytoprotective effects of F16bP have been documented in lesions caused by chemicals or cold storage, in a decrease in mortality during sepsis shock and even in the prevention of bone loss in experimental osteoporosis. Intriguingly, protection in such a variety of targets and animal models suggests that the mechanisms induced by F16bP are complex and involve different pathways. In this review we will discuss the most recent theories concerning the molecular model of action of F16bP inside cells. These include its incorporation as an energy substrate, the mechanism for the improvement of ATP availability, and for preservation of organelle membrane stability and functionality. In addition we will present new evidences regarding the capacity of F16bP to decrease oxidative stress by limiting free radical production and improving antioxidant systems, including the role of nitric oxide in the protective mechanism induced by F16bP. Finally we will review the proposed mechanisms for explaining its anti-inflammatory, immunomodulatory and neuroprotective properties.

This study investigated the treatment effects of a new compound, strontium fructose 1, 6-diphosphate (FDP-Sr), in cyclophosphamide (CP)-induced oligozoospermia. FDP-Sr, with extra high-energy supply, could reverse male hypogonadism in the testis. Male Wistar rats were randomly divided into three groups: control group (vehicle treated), CP group and CP + FDP-Sr group. Both CP group and CP + FDP-Sr groups were orally administered CP (20 mg kg(-1) ) consecutively for the first 7 days to establish CP-induced testicular toxic models. Subsequently, CP group was given orally distilled water per day, whereas CP + FDP-Sr group was received FDP-Sr (200 mg kg(-1) ) for 49 days. Compared to the CP group, the FDP-Sr group showed significantly increased levels of serum testosterone, testis relative weights and epididymal sperm counts in rats. In addition, rats treated by FDP-Sr showed the recuperative activities of testicular marker enzymes and normalised levels of antioxidants in tissue. Testicular protection of FDP-Sr was further demonstrated by enhancing expression of P450scc, reducing ability of FAS/FASL and generating cytoprotection in the histopathological study. FDP-Sr appeared to possess an ability to attenuate CP-induced reproduction toxicity via the activation of antioxidants and steroidogenesis enzymes, and alleviate oligozoospermia via inhibition of testicular apoptosis by FAS/FASL pathway., (© 2014 Blackwell Verlag GmbH.)

Fructose 1,6-bisphosphate (FBP) is an endogenous intermediate of the glycolytic pathway. Exogenous administration of FBP has been shown to exert protective effects in a variety of ischemic injury models, which are attributed to its ability to sustain glycolysis and increase ATP production. Here, we demonstrated that a single treatment with FBP markedly attenuated arthritis, assessed by reduction of articular hyperalgesia, joint swelling, neutrophil infiltration and production of inflammatory cytokines, TNF and IL-6, while enhancing IL-10 production in two mouse models of arthritis. Our mechanistic studies showed that FBP reduces joint inflammation through the systemic generation of extracellular adenosine and subsequent activation of adenosine receptor A2a (A2aR). Moreover, we showed that FBP-induced adenosine generation requires hydrolysis of extracellular ATP through the activity of the ectonucleosides triphosphate diphosphohydrolase-1 (ENTPD1, also known as CD39) and ecto-5'-nucleotidase (E5NT, also known as CD73). In accordance, inhibition of CD39 and CD73 abolished anti-arthritic effects of FBP. Taken together, our findings provide a new insight into the molecular mechanism underlying the anti-inflammatory effect of FBP, showing that it effectively attenuates experimental arthritis by activating the anti-inflammatory adenosinergic pathway. Therefore, FBP may represent a new therapeutic strategy for treatment of rheumatoid arthritis (RA).

Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with salutary effects in various brain injury models, but its neuroprotective mechanism is incompletely understood. In this study, we examined the effects of FBP on the expression of adhesion molecules in cerebrovascular endothelial cells and explored the possible mechanisms therein involved. FBP significantly down-regulated lipopolysaccharide (LPS)-induced expression of adhesion molecules and leukocyte adhesion to brain endothelial cells and inhibited NF-κB activity, which is implicated in the expression of adhesion molecules. FBP abrogated ICAM-1 expression and NF-κB activation induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or overexpression of MyD88 or TRAF6. FBP suppressed TRAF6-induced phosphorylation of TAK1, IKKβ and IκBα, but fail to affect NF-κB activity induced by ectopic expression of IKKβ. In addition, LPS-induced IRAK-1 phosphorylation was inhibited by FBP, suggesting the presence of multiple molecular targets of FBP in MyD88-dependent signaling pathway. FBP significantly attenuated ICAM-1 expression and NF-κB activity induced by poly[I:C] or overexpression of TRIF or TBK1. FBP significantly repressed the expression of interferon-β (IFN-β) and the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C] or overexpression of TRIF or TBK1, but fail to affect IRF3 activity induced by ectopic expression of constitutively active IRF3. Overall, our results demonstrate that FBP modulates both MyD88- and TRIF-dependent signaling pathways of TLR4 and subsequent inflammatory responses in brain endothelial cells, providing insight into its neuroprotective mechanism in brain injury associated with inflammation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study aims to investigate the apoptosis-inducing effect of fructose 1,6-bisphosphate (F1,6BP) on the related mechanism of papillary thyroid carcinoma W3 and T cells. W3 cells were treated with F1,6BP alone or in combination with antioxidant catalase (CAT) or N-acetyl-L-cysteine (NAC). The changes of cell viability and cell nucleus morphology were examined by cell proliferation assay and Hoechst staining, and apoptosis levels of these cells were measured with flow cytometry. The changes of reactive oxygen species (ROS) level and the percentage of oxidized glutathione in total glutathione in W3 cells were detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining or colorimetry assay. At the same time, real-time fluorescence quantitative PCR was adopted to evaluate the expression levels of CAT and glutathione peroxidase (GSH-Px) mRNAs in W3 cells. F1,6BP inhibited the growth of W3 cells significantly, coupling with an increase in intracellular ROS level and the percentage of oxidized glutathione in total glutathione. Typical apoptotic morphological changes of the cell nucleus happened. The apoptosis rate and GSH-Px and CAT mRNAs expression levels were upregulated after F1,6BP treatment. The antitumor effect of F1,6BP was significantly decreased after W3 cells were pretreated with NAC and CAT. F1,6BP can induce the apoptosis of W3 cells through upregulating the generation of ROS, especially the production of H2O2.

The accumulation of advanced oxidation protein products (AOPPs) has been linked to several pathological conditions, and their levels are formed during oxidative stress as a result of reactions between plasma proteins and chlorinated oxidants produced by myeloperoxidase (MPO). However, it was suggested that the generation of this mediator of inflammation may also occur via an MPO-independent pathway. The aim of this study was to induce the formation of AOPPs in vitro through Fenton reaction and to investigate whether this generation could be counteracted by N-acetylcysteine (NAC) and fructose-1,6-bisphosphate (FBP). The complete Fenton system increased the AOPPs levels and both NAC and FBP were capable of inhibiting the formation of Fenton reaction-induced AOPPs. These data provide a new hypothesis about another pathway of AOPPs formation, as well as report that NAC and FBP may be good candidates to neutralize pro-inflammatory and pro-oxidant effects of AOPPs in several diseases.

We aimed to investigate the antitumor effect and mechanism of fructose 1,6-bisphosphate (F1,6BP) in a hepatocellular carcinoma cell line. HepG2 cells were treated with different concentrations of F1,6BP alone or in combination with antioxidant N-acetyl-L-cysteine (NAC) or catalase (CAT), and cell proliferation assays were performed. Nuclear morphology was observed by fluorescence microscopy after Hoechst staining, and apoptosis was measured with flow cytometry. Changes in reactive oxygen species (ROS) levels in HepG2 cells were detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. A colorimetric assay was adopted to determine the percentage of oxidized glutathione in these cells. CAT and glutathione peroxidase (GSH-Px) mRNA expression levels in HepG2 cells were measured by real-time fluorescence quantitative PCR. HepG2 cell proliferation was significantly inhibited by F1,6BP, accompanied by an increase in intracellular ROS levels and oxidized glutathione. Upregulated apoptosis and characteristic nuclear morphological changes were observed, and the expression of CAT and GSH-Px mRNA was increased after F1,6BP treatment. The antitumor effect of F1,6BP was significantly decreased after pretreatment with NAC and CAT in HepG2 cells. In conclusion, F1,6BP can induce the apoptosis of HepG2 cells. The mechanism involved may be associated with the generation of ROS, especially the production of H2O2.

Hepatic stellate cells (HSC) play a key role in liver fibrogenesis. Activation of PPARγ and inhibition of fibrogenic molecules are potential strategies to block HSC activation and differentiation. Aware that the process of hepatic fibrosis involves inflammatory mediators, various anti-inflammatory substances have been studied in an attempt to revert fibrosis. The purpose of this study was to investigate the in vitro effects of fructose-1,6-bisphosphate (FBP) on HSC phenotype reversion. The results demonstrated that FBP induced quiescent phenotype in GRX cells via PPARγ activation. Significant decrease in type I collagen mRNA expression was observed in the first 24h of treatment. These events preceded the reduction of TGF-β1 and total collagen secretion. Thus, FBP promoted downregulation of HSC activation by its antifibrotic action. These findings demonstrate that FBP may have potential as a novel therapeutic agent for the treatment of liver fibrosis., (© 2013 Elsevier B.V. All rights reserved.)

A novel strontium salt compound strontium fructose 1, 6-diphosphate (FDP-Sr) has been proved to have highly effective for bone loss via dual effects of stimulating bone formation and suppressing bone absorption. In the present study, metabolomic approach was used to identify and study potential biomarkers associated with the effect and safety of FDP-Sr. The metabolomic profiles of bone loss induced by estrogen deficiency in a rat model was described to attain a system-level map of the shift on the metabolic response in plasma using GC/TOF-MS, after FDP-Sr was orally administered at the dose of 110 mg/kg/day for the prevention and 220 mg/kg/day for the treatment. Meanwhile, bone turnover biomarkers and bone mineral density were investigated to identify the specific changes of potential anti-osteoporosis effects of FDP-Sr. The differences in metabolic profiles between osteoporosis rats and FDP-Sr treated rats were well observed by the partial least squares-discriminant analysis (PLS-DA) to the MS spectra. Some metabolites including homocysteine, arachidonic acid, alanine, and hydroxyproline, which significantly changed during osteoporosis progression could be effectively reversed after FDP-Sr therapy. Of course some metabolites such as uric acid, glyceric acid, octadecadienoic acid, docosahexaenoic acid, oleic acid, and hexadecanoic acid were not found to reverse significantly after FDP-Sr administration. These results delineated the FDP-Sr effects-related metabolic alterations in the bone loss rats, suggesting that metabonomic analysis could provide helpful information on the new potential biomarkers relating to the mechanism of anti-osteoporosis action and side effects of FDP-Sr against estrogen deficiency induced bone loss., (© 2013 Elsevier B.V. All rights reserved.)

Fructose-1,6-bisphosphate (FBP), a glycolytic intermediate, has neuroprotective effects in various brain injury models. However, its effects on blood-brain barrier (BBB) are largely unknown. In this study, we investigated the effects of FBP on lipopolysaccharide (LPS)-induced BBB dysfunction in in vitro BBB model comprising co-culture of mouse brain endothelial cell line, bEnd.3 and mouse primary astrocyte and explored its action mechanism therein involved. LPS induced the impairment of endothelial permeability and transendothelial electrical resistance (TEER). The functional changes were confirmed by alterations in immunostaining for junctional proteins occludin, ZO-1 and VE-cadherin, such as the loss of cortical staining pattern and appearance of intercellular gaps in endothelial cells. Co-administration of FBP alleviated the deleterious effects of LPS on BBB permeability and TEER in a dose dependent manner. And also FBP inhibited the LPS-induced changes in the distribution of endothelial junctional proteins, resulting in the better preservation of monolayer integrity. FBP suppressed the production of reactive oxygen species (ROS) but did not affect cyclooxygenase-2 expression and prostaglandin E₂ production in endothelial cells stimulated with LPS. Taken together, these data suggest that FBP could ameliorate LPS-induced BBB dysfunction through the maintenance of junctional integrity, which might be mediated by downregulation of ROS production.

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.

Fructose 1,6-bisphosphate (F1,6BP) has been widely used as a therapeutic agent for different harmful conditions in a variety of tissues. The hypothesis of the present work was that the increase in nitric oxide production and the prevention of oxidative stress induced by exogenous F1,6BP mediate its protective effect against the hepatotoxic action of GalN. Experimental groups used were sham, F1,6BP (2g/kg bw i.p.), GalN (0.4g/kg bw i.p), l-NAME (10mg/kg bw i.v.), F1,6BP+GalN, l-NAME+GalN and l-NAME+F1,6BP+GalN. Animals were killed after 24h of bolus administration. F1,6BP induced an increase in NO and the redox ratio (GSH/GSSG) in liver. Western blot assays pointed to overexpression of liver eNOS in F1,6BP-treated rats. The hepatic injury induced by GalN increased transaminases in plasma and decreased the reduced/oxidized glutathione ratio in liver. The concomitant administration of F1,6BP reversed this damage, while the addition of l-NAME worsened the liver injury. We provided evidence that this F1,6BP-induced protection may be related to the increase in NO production through the positive modulation of eNOS, and the increase in intracellular reduced glutathione, thus providing a higher reducing capacity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Fructose-1,6-diphosphate (FDP) is a metabolite in the glycolytic pathway created from glucose. Exogenously administered FDP increases the yield of ATP from anaerobic glycolysis. FDP reduces ischaemic tissue area in experimentally-induced cerebral and myocardial infarction and improves haemodynamics post-cardiac bypass. We hypothesised that FDP improves haemodynamics in propranolol and verapamil poisoning., Method: Anesthetized Wistar rats were instrumented to record BP, heart rate (HR), cardiac output (CO) and QRS-duration. Propranolol or verapamil were infused continually. When BP dropped by 50%, propranolol-poisoned rats received one of 10% FDP125 mg/kg or 10% FDP250 mg/kg loading dose over 20 minutes followed by infusion 20 mg/kg/h. Verapamil-poisoned rats received the higher dosing regimen of FDP250. Controls received comparable volumes of 10% glucose. Haemodynamic time-points were compared for FDP to control by unpaired t-test or Mann-Whitney test as appropriate (p <  0.05). Survival was assessed using Kaplan-Meier survival analysis., Results: FDP-treated animals survived significantly longer than glucose-treated controls at both doses in propranolol poisoning and in verapamil-poisoning. In propranolol poisoning, FDP250-treated animals showed a statistically significant increase in BP. However, there was no significant difference in cardiac output at this dose. There were also no significant differences in any haemodynamic parameters compared to control at the lower FDP dose in propranolol poisoning or in verapamil poisoning., Conclusion: FDP improved survival for both toxicants with an improvement in haemodynamics at the higher dose in propranolol poisoning. Future research could examine the efficacy of FDP in other beta-blocker and calcium channel-blocker poisoning as well as in concert with established inotropic therapies in drug-induced cardiovascular collapse.

Due to the fact that an increased number of patients have experienced bloodstream infections caused by Candida species and the high mortality of this infection, there is a need for a strategy to reduce this scenery. One possible strategy is the use of new drugs, such as fructose-1,6-bisphosphate (FBP), which is a high-energy glycolytic metabolite and has shown to have therapeutic effects in several pathological conditions such as ischemia, shock, toxic injuries, and bacterial sepsis. The aim of this manuscript was to determine the role of FBP in experimental Candida albicans bloodstream infection. We used mice that were divided into three experimental groups: sham (not induced), bloodstream infection (induced with intratracheal instillation of C. albicans) and FBP (bloodstream infection plus FBP 500 mg/kg i.p.). Blood was taken for assessment of complete hematological profile and cytokine assay (IL-6 and MCP-1). Results of the study demonstrated that mortality decreased significantly in groups that received FBP. All cytokine and hematological indexes of FBP group were similar to bloodstream infection group with exception of platelets count. FBP significantly prevented the decrease in platelets. Taken together, our results demonstrate that FBP prevented the mortality in C. albicans bloodstream infection.

It has been previously showed that fructose-1,6-bisphosphate (FBP) has anti-inflammatory and immunomodulatory effects on several experimental inflammation models. However, the effects and mechanism of FBP on Zymosan-induced acute lung injury (ALI) in mice had not been tested. In this study, our aim was to assess the anti-inflammatory activities of FBP on Zymosan-induced ALI. We found that in vivo treatment with FBP (500 mg/kg i.p.) markedly decreased the nitric oxide (NO) levels in the lungs and significantly reduced bronchoalveolar lavage fluid total cell and neutrophil counts and protein exudation after Zymosan challenge. Furthermore, FBP inhibited inducible nitric oxide synthase (iNOS) activities in RAW macrophages. Meanwhile, FBP did not inhibit the cyclooxigenase 2, interleukin-6, and nuclear factor kappa B transcription. Taken together, these results suggest that FBP shows anti-inflammatory effects through inhibiting lung edema, NO, and iNOS activities.

In this study, we evaluated the neurotrophic effects of magnesium fructose 1, 6-diphosphate (FDP-Mg) on cortical neurons. The results demonstrated that FDP-Mg promoted dendrite outgrowth and neuronal survival in a dose-dependent manner. In order to investigate the associated mechanisms, we determined adenosine triphosphate (ATP) levels and brain-derived neurotrophic factor (BDNF) mRNA expression in cortical neurons. Treatment with FDP-Mg significantly increased ATP levels and BDNF mRNA expression in cortical neurons. These data suggest that FDP-Mg can exert neurotrophic effects on cortical neurons. The increases in BDNF mRNA expression and cellular ATP levels are involved in the neurotrophic effects produced by FDP-Mg.

Anapleurosis is the filling of the tricarboxylic acid cycle with four-carbon units. The common substrate for both anapleurosis and glucose phosphorylation in bacteria is the terminal glycolytic metabolite phosphoenolpyruvate (PEP). Here we show that Escherichia coli quickly and almost completely turns off PEP consumption upon glucose removal. The resulting buildup of PEP is used to quickly import glucose if it becomes available again. The switch-like termination of anapleurosis results from depletion of fructose-1,6-bisphosphate (FBP), an ultrasensitive allosteric activator of PEP carboxylase. E. coli expressing an FBP-insensitive point mutant of PEP carboxylase grow normally when glucose is steadily available. However, they fail to build up PEP upon glucose removal, grow poorly when glucose availability oscillates and suffer from futile cycling at the PEP node on gluconeogenic substrates. Thus, bacterial central carbon metabolism is intrinsically programmed with ultrasensitive allosteric regulation to enable rapid adaptation to changing environmental conditions.

Aim: To evaluate the protective effects of strontium fructose 1,6-diphosphate (FDP-Sr), a novel strontium salt that combined fructose 1,6-diphosphate (FDP) with strontium, on bone in an ovariectomy-induced model of bone loss., Methods: Eighty female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated. Three months later, the rats were assigned to six groups (10 for each): sham-operated, OVX control, OVX+FDP-Sr (110, 220, or 440 mg/kg), or OVX+strontium ranelate (SR, 180 mg/kg). Drugs were administered orally for 3 months. When the treatment was terminated, the following parameters were assessed: bone mineral density (BMD), the biomechanical properties of the femur and lumbar vertebrae, trabecular histomorphology, serum phosphorus, calcium, bone-specific alkaline phosphatase (B-ALP), tartrate-resistant acid phosphatase 5b (TRACP5b), N-telopeptide of type I collagen (NTx) and a series of markers for oxidative stress. Receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) levels in serum were measured using ELISA and their gene expression levels in the bone were measured using R-T PCR., Results: Treatment with FDP-Sr (220 or 440 mg/kg) or SR (180 mg/kg) significantly increased the BMD and improved the bone microarchitecture and bone strength in OVX rats. The treatments also decreased in the levels of H(2)O(2) and MDA, restored the CAT level in serum and bone marrow, increased the serum B-ALP and decreased NTx and TRACP 5b in OVX rats. Treatment with FDP-Sr decreased the RANKL level, and increased the OPG level in serum in a dose-dependent manner. It also significantly down-regulated the RANKL expression and up-regulated OPG expression in bone marrow., Conclusion: FDP-Sr may be an effectve treatment for postmenopausal osteoporosis that acts, in part, via a decrease in osteoclastogenesis through the OPG\RANKL\RANK pathway.

Phosphofructokinase (PFK-1) activity was examined in L(3) and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4±0.2 to 1.7±0.2 in L(3)s and 2.0±0.3 to 2.4±0.4 in adults) and the K(½) for fructose 6 phosphate (from 0.35±0.02 to 0.75±0.05mM in L(3)s and 0.40±0.03 to 0.65±0.05mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with neuroprotective action in various brain injury models. However, the mechanism underlying the neuroprotection of FBP has not been fully defined. In this study, we investigated whether FBP inhibits endotoxin-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in microglial cells and explored the possible mechanisms of the effects of FBP., Main Methods: Murine microglial cell line BV2 and primary cultured murine microglial cells were used. NO production and iNOS expression were determined by Griess reaction, RT-PCR and Western blot. Luciferase assay using iNOS promoter-luciferase (iNOS-Luc) construct was adopted for measuring transcriptional activity., Key Findings: FBP dose-dependently suppressed lipopolysaccharide (LPS)-induced NO production, along with reducing the expression of iNOS at both the protein and mRNA level in primary cultured murine microglia and BV2 cells. FBP significantly inhibited iNOS promoter activity but stabilized iNOS mRNA. Among transcription factors known to be related to iNOS expression, activator protein (AP-1) activation was significantly blocked by FBP. FBP suppressed LPS-induced phosphorylation of three MAPK subtypes-p38 MAPK, JNK and ERK. FBP inhibited LPS-induced production of reactive oxygen species (ROS) and decreased intracellular GSSG/GSH ratio., Significance: Our findings suggest that FBP attenuates the LPS-induced iNOS expression through inhibition of JNK and p38 MAPK, which might be related to ROS downregulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objectives: Phosphodiesterase type 5 inhibitors are potent in relieving erectile dysfunction (ED), however, they are less satisfactory in diabetic patients, probably due to the pro-inflammatory biomarkers caused by diabetes. Therefore, it was interesting to compare the effects of sildenafil with strontium fructose 1,6-diphosphate (FDP-Sr) on cavernosal vascular activity and expressions of pro-inflammatory biomarkers in diabetic rats., Methods: Male Sprague-Dawley rats were injected with streptozocin (60 mg/kg, i.p.) to develop diabetes. The animals were diabetic for eight weeks with sildenafil (12 mg/kg per day) or FDP-Sr (200 mg/kg per day) being administered for the last four of those eight weeks., Key Findings: Sildenafil was more effective in relieving reduced vascular dilatation (relevant to ED), but less in attenuating over-expressions of NADPH oxidase p22, p47 and p67 subunits, and ET(A/B) R (endothelin receptor type A and type B) in the diabetic cavernosum. In contrast, FDP-Sr was less effective in improving ED, but more effective in normalizing the abnormal NADPH oxidase and ET(A/B) R., Conclusions: The activated NADPH oxidase and upregulated ET(A) R and ET(B) R due to diabetic lesions played a minor or moderate role in ED. By offering extra ATP, FPD-Sr suppressed these abnormalities, however, sildenafil did not. A combined therapy of sildenafil with FDP-Sr may be more effective in relieving ED in diabetic patients through normalizing pro-inflammatory cytokines and improving the nitric oxide/cGMP pathway in the cavernosum., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Identification of allosteric binding site modulators have gained increased attention lately for their potential to be developed as selective agents with a novel chemotype and targeting perhaps a new and unique binding site with probable fewer side effects. Erythrocyte pyruvate kinase (R-PK) is an important glycolytic enzyme that can be pharmacologically modulated through its allosteric effectors for the treatment of hemolytic anemia, sickle-cell anemia, hypoxia-related diseases, and other disorders arising from erythrocyte PK malfunction. An in-silico screening approach was applied to identify novel allosteric modulators of pyruvate kinase. A small-molecules database of the National Cancer Institute (NCI), was virtually screened based on structure/ligand-based pharmacophore. The virtual screening campaign led to the identification of several compounds with similar pharmacophoric features as fructose-1,6-bisphosphate (FBP), the natural allosteric activator of the kinase. The compounds were subsequently docked into the FBP-binding site using the programs FlexX and GOLD, and their interactions with the protein were analyzed with the energy-scoring function of HINT. Seven promising candidates were obtained from the NCI and subjected to kinetics analysis, which revealed both activators and inhibitors of the R-isozyme of PK (R-PK).

Background and Aims: Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis., Materials and Methods: Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW+SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor., Results: AST was significantly lower during cold storage with UW than with the older media (P=.001); ALT was lower in the FBP group (P=.023) and LDH was lower in the FBP and SNAC groups (P=.007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups (P=.008 and P=.006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 (P=.004). Renin levels were significantly lower in the FBP group (P=.022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor., Conclusion: In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD+-dependent L-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator D-fructose-1,6-bisphosphate (Fru-1,6-P2). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with Vmax for the reduction of pyruvate being approximately tenfold that of the value for L-lactate oxidation at pH 7.5. No deviation from Michaelis-Menten kinetics was observed in the presence of Fru-1,6-P2, while a sigmoidal response (indicative of positive cooperativity) was seen towards L-lactate without Fru-1,6-P2. Strikingly, when introduced into an lldD- strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on L-lactate as the sole carbon source.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.

Fructose 1,6 biphosphate (F1,6BP) exerts a protective effect in several in vitro models of induced injury and in isolated organs; however, few studies have been performed using in vivo hypothermia. Here we studied the effects of deep hypothermia (21ºC) and rewarming in anaesthetised rats after F1,6BP administration (2 g/kg body weight). Acid-base and oxidative stress parameters (plasma malondialdehyde and glutathione, and erythrocyte antioxidant enzymes) were evaluated. Erythrocyte and leukocyte numbers in blood and plasma nitric oxide were also measured 3 h after F1,6BP administration in normothermia animals. In the absence of F1,6BP metabolic acidosis developed after rewarming. Oxidative stress was also evident after rewarming, as shown by a decrease in thiol groups and in erythrocyte superoxide dismutase, catalase and GSH-peroxidase, which corresponded to an increase in AST in rewarmed animals. These effects were reverted in rats treated with F1,6BP. Blood samples of F1,6BP-treated animals showed a significant increase in plasma nitric oxide 3 h after administration, coinciding with a significant rise in leukocyte number. F1,6BP protection may be due to the decrease in oxidative stress and to the preservation of the antioxidant pool. In addition, we propose that the reduction in extracellular acidosis may be due to improved tissue perfusion during rewarming and that nitric oxide may play a central role., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hypothermic preservation of bioartificial liver (BAL) has long been appreciated in BAL storage and transportation. However, the deterioration of cell activity during hypothermia/rewarming limits its clinical use and the complete prevention of hypothermia-induced hepatocyte injury has not been achieved. In this article, a miniaturized BAL that underwent three preservation stages (i.e. pre-incubation, hypothermia and rewarming) was applied as a hypothermic preservation model to locate the protection of several protective agents against hypothermia-induced cell injury. The agents, including vitamin E, schisandrin B, glycyrrhizic acid, N-acetyl-cysteine, ruthenium red, trehalose, anisodamine, fructose-1, 6-diphosphate, cyclosporin A and matrine (Mat), were found to exert their functions at different preservation stages, which were speculated to associate with the specific protection of each agent as well as the corresponding cell injuries at each stage. Such hypothesis was further strengthened by focusing on Mat, which only suppressed the hypothermia-induced injury through the inhibition of Ca(2+) overload at the rewarming stage, whereas its presence at the hypothermic stage excessively down-regulated the cytosolic free Ca(2+) and then aggravated cell death. The results indicate that the specific cell injury at each preservation stage requires a corresponding protective agent. However, the untimely misuse of the agents may inversely aggravate cell injury.

Background: Fructose 1,6-biphosphate (FBP) has been shown to exert therapeutic effects in models of ischemia-reperfusion in organs other than the liver. This study compared FBP and University of Wisconsin (UW) solution during cold storage and reperfusion, among mitochondria of adult male Wistar rat livers., Methods: Adult male Wistar rats were assigned to two groups according to the preservation solution used; UW or FBP Aspartate transaminase (AST), alanine transferase (ALT); and lactic dehydrogenase (LDH) were measured in samples of the storage solution obtained at 2, 4 and 6 hours of preservation. After 6 hours of cold storage, we reperfused the liver, taking blood samples to measure AST, ALT, LDH, and throbarbituric acid reactive substances (TBARS). Hepatic fragments were processed for histologic analysis; for determinations of TBARS, catalase, and nitric oxide as well as for mitochondrial evaluation by infrared spectroscopy., Results: During cold preservation, levels of AST and LDH in the storage solution were lower among the FBP group, but after reperfusion, serum levels of AST, ALT, and LDH were higher in this group, as was catalase activity. TBARS and nitric oxide were comparable between the groups. In the UW group there was a higher amide I/amide II ratio than in the FBP group, suggesting an abnormal protein structure of the mitochondrial membrane. No signs of preservation injury were observed in any liver biopsy, but sinusoidal congestion was present in livers preserved with FBP., Conclusion: FBP showed a protective effect for preservation during cold storage seeming to protect the mitochondrial membrane although it did not prevent reperfusion injury., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Human cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved., Methodology/principal Findings: In the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux., Conclusions/significance: Posttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells.

Cisplatin is one of the most active cytotoxic agents in the treatment of cancer, but its clinical use is frequently limited by nephrotoxicity. The study presented here attempted to evaluate the effect of fructose-1,6-bisphosphate in the cisplatin-induced nephrotoxicity in rats. The drugs were administered intraperitoneally as a single dose: sodium chloride 0.9%, cisplatin (6 mg/kg), fructose-1,6-bisphosphate (500 mg/kg), and cisplatin plus fructose-1,6-bisphosphate (6 and 500 mg/kg, respectively). The use of cisplatin resulted in significant elevation of serum creatinine and urea. The group that received cisplatin plus fructose-1,6-bisphosphate presented a significantly lower level of creatinine and urea compared to the cisplatin group. Acute tubular necrosis was demonstrated in the animals that received cisplatin and a less severe one in the cisplatin plus fructose-1,6-bisphosphate group. Fructose-1,6-bisphosphate has a protective effect over renal function and renal parenchyma in a rat experimental model of cisplatin-induced nephrotoxicity. The anti-inflammatory effect of fructose-1,6-bisphosphate confirms its protective effect in cases of cellular injury.