Tetramerization of pyruvate kinase M2 attenuates graft-versus-host disease by inhibition of Th1 and Th17 differentiation.

Lethal graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem-cell transplantation (Allo-HSCT). Pyruvate kinase M2 (PKM2) is essential for CD4+ T-cell differentiation. Using the well-characterized mouse models of Allo-HSCT, we explored the effects of TEPP-46-induced PKM2 tetramerization on GVHD and graft-versus-leukemia (GVL) activity. TEPP-46 administration significantly improved the survival rate of GVHD. The severity of GVHD and histopathological damage of GVHD-targeted organs were obviously alleviated by PKM2 tetramerization. Additionally, tetramerized PKM2 inhibited the activation of NF-κB pathway and decreased the inflammation level of GVHD mice. PKM2 tetramerization blocked Th1 and Th17 cell differentiation and secretion of pro-inflammatory cytokine (IFN-γ, TNF-α, and IL-17). Meanwhile, differentiation of Treg cells and IL-10 secretion were promoted by tetramerized PKM2. These findings demonstrated that PKM2 enhanced the augment of Th1 and Th17 cells to accelerate the progression of GVHD, and allosteric activation of PKM2 targeted Th1 and Th17 cells attenuated GVHD. Furthermore, we also confirmed that TEPP-46 administration did not compromise GVL activity and resulted in slightly improvement of leukemia-free survive. Thus, targeting Th1 and Th17 cell response with PKM2 allosteric activator may be a promising therapeutic strategy for GVHD prevention while preserving the GVL activity in patients receiving Allo-HSCT.

Metformin Increases the Response of Cholangiocarcinoma Cells to Gemcitabine by Suppressing Pyruvate Kinase M2 to Activate Mitochondrial Apoptosis.

BACKGROUND: Cholangiocarcinoma (CCA) is a malignant tumor with a high mortality rate. Resistance to chemotherapy remains a major challenge related to cancer treatment, and increasing the sensitivity of cancer cells to therapeutic drugs is a major focus of cancer treatment. AIMS: We purposed to explore the role of Metformin in CCA involved in chemotherapeutic sensitivity and Pyruvate kinase M2 (PKM2) through regulating mitochondrial apoptosis in the present study. METHODS: CCA cell lines of HCC9810 and RBE were treated with Metformin companied with antagonists or agonists of PKM2, cells sensitivity to Gemcitabine, cell migration and invasion along with apoptosis, which is mediated by JC-1 and LDH were assayed. RESULTS: Our results indicated that Metformin and Gemcitabine exhibit synergistic effect on inhibition of cholangiocarcinoma cell viability, cell migration and invasion as well as promotion apoptosis of cholangiocarcinoma cells. In vivo, Metformin combined with Gemcitabine has cooperation in inhibiting the growth of cholangiocarcinoma cell-derived tumors. Moreover, Metformin and Gemcitabine inhibited expression of PKM2 and PDHB in HCC9810 and RBE. CONCLUSION: Our study suggested that Metformin may increase the response of cholangiocarcinoma cells to Gemcitabine by suppressing PKM2 to activate mitochondrial apoptosis.

KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production.

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (KATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited KATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit KATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP . In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on KATP activity. Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.

Design, synthesis and biological evaluation of novel modified dual-target shikonin derivatives for colorectal cancer treatment.

Warburg effect provides energy and material essential for tumor proliferation, the reverse of Warburg effect provides insights into the development of a novel anti-cancer strategy. Pyruvate kinase 2 (PKM2) and pyruvate dehydrogenase kinase 1 (PDK1) are two key enzymes in tumor glucose metabolism pathway that not only contribute to the Warburg effect through accelerating aerobic glycolysis, but also serve as druggable target for colorectal cancer (CRC). Considering that targeting PKM2 or PDK1 alone does not seem to be sufficient to remodel abnormal glucose metabolism and achieve significant antitumor activity, a series of novel benzenesulfonyl shikonin derivatives were designed to regulate PKM2 and PDK1 simultaneously. By means of molecular docking and antiproliferative screen, we found that compound Z10 could act as the combination of PKM2 activator and PDK1 inhibitor, thereby significantly inhibited glycolysis that reshaping tumor metabolism. Moreover, Z10 could inhibit proliferation, migration and induce apoptosis in CRC cell HCT-8. Finally, the in vivo anti-tumor activity of Z10 was evaluated in a colorectal cancer cell xenograft model in nude mice and the results demonstrated that Z10 induced tumor cell apoptosis and inhibited tumor cell proliferation with lower toxicity than shikonin. Our findings indicated that it is feasible to alter tumor energy metabolism through multi-target synergies, and the dual-target benzenesulfonyl shikonin derivative Z10 could be a potential anti-CRC agent.

In vitro galactose impairs energy metabolism in the brain of young rats: protective role of antioxidants.

We, herein, investigated the in vitro effects of galactose on the activity of pyruvate kinase, succinate dehydrogenase (SDH), complex II and IV (cytochrome c oxidase) of the respiratory chain and Na+K+-ATPase in the cerebral cortex, cerebellum and hippocampus of 30-day-old rats. We also determined the influence of the antioxidants, trolox, ascorbic acid and glutathione, on the effects elicited by galactose. Galactose was added to the assay at concentrations of 0.1, 3.0, 5.0 and 10.0 mM. Control experiments were performed without galactose. Galactose, at 3.0, 5.0 and 10.0 mM, decreased pyruvate kinase activity in the cerebral cortex and at 10.0 mM in the hippocampus. Galactose, at 10.0 mM, reduced SDH and complex II activities in the cerebellum and hippocampus, and reduced cytochrome c oxidase activity in the hippocampus. Additionally, decreased Na+K+-ATPase activity in the cerebral cortex and hippocampus; conversely, galactose, at 3.0 and 5.0 mM, increased this enzyme's activity in the cerebellum. Data show that galactose disrupts energy metabolism and trolox, ascorbic acid and glutathione addition prevented the majority of alterations in the parameters analyzed, suggesting the use of antioxidants as an adjuvant therapy in Classic galactosemia.

Reduced CircSMOC1 Level Promotes Metabolic Reprogramming via PTBP1 (Polypyrimidine Tract-Binding Protein) and miR-329-3p in Pulmonary Arterial Hypertension Rats.

BACKGROUND: Pulmonary arterial hypertension maintains rapid cell proliferation and vascular remodeling through metabolic reprogramming. Recent studies suggested that circRNAs play important role in pulmonary vascular remodeling and pulmonary arterial smooth muscle cells proliferation. However, the relationship between circRNA, cell proliferation, and metabolic reprogramming in pulmonary arterial hypertension has not been investigated. METHODS: RNA-seq and qRT-PCR reveal the differential expression profile of circRNA in pulmonary arteries of pulmonary arterial hypertension rat models. Transfection was used to examine the effects of circSMOC1 on pulmonary artery smooth muscle cells, and the roles of circSMOC1 in vivo were investigated by adenoassociated virus. Mass spectrometry, RNA pull-down, RNA immunoprecipitation, and dual-luciferase reporter assay were performed to investigate the signaling pathway of circSMOC1 regulating the metabolic reprogramming. RESULTS: CircSMOC1 was significantly downregulated in pulmonary arteries of pulmonary arterial hypertension rats. CircSMOC1 knockdown promoted proliferation and migration and enhanced aerobic glycolysis of pulmonary artery smooth muscle cells. CircSMOC1 overexpression in vivo alleviates pulmonary vascular remodeling, right ventricular pressure, and right heart hypertrophy. In the nucleus, circSMOC1 directly binds to PTBP1 (polypyrimidine tract-binding protein), competitively inhibits the specific splicing of PKM (pyruvate kinase M) premRNA, resulting in the upregulation of PKM2 (pyruvate kinase M2), the key enzyme of aerobic glycolysis, to enhance glycolysis. In the cytoplasm, circSMOC1 acted as a miR-329-3p sponge, and its reduction in pulmonary arterial hypertension suppressed PDHB (pyruvate dehydrogenase E1 subunit beta) expression, leading to the impairment of mitochondrial oxidative phosphorylation. CONCLUSIONS: circSMOC1 is crucially involved in the metabolic reprogramming of pulmonary artery smooth muscle cells through PTBP1 and miR-329-3p to regulate pulmonary vascular remodeling in pulmonary arterial hypertension.

AMPK's double-faced role in advanced stages of prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer deaths in men. Unfortunately, a very limited number of drugs are available for the relapsed and advanced stages of PCa, adding only a few months to survival; therefore, it is vital to develop new drugs. 5´ AMP-activated protein kinase (AMPK) is a master regulator of cell metabolism. It plays a significant role in the metabolism of PCa; hence, it can serve well as a treatment option for the advanced stages of PCa. However, whether this pathway contributes to cancer cell survival or death remains unknown. The present study reviews the possible pathways by which AMPK plays role in the advanced stages of PCa, drug resistance, and metastasis: (1) AMPK has a contradictory role in promoting glycolysis and the Warburg effect which are correlated with cancer stem cells (CSCs) survival and advanced PCa. It exerts its effect by interacting with hypoxia-induced factor 1 (HIF1) α, pyruvate kinase 2 (PKM2), glucose transporter (GLUT) 1 and pyruvate dehydrogenase complex (PDHC), which are key regulators of glycolysis; however, whether it promotes or discourage glycolysis is not conclusive. It can also exert an anti-CSC effect by negative regulation of NANOG and epithelial-mesenchymal transition (EMT) transcription factors, which are the major drivers of CSC maintenance; (2) the regulatory effect of AMPK on autophagy is also noticeable. Androgen receptors' expression increases AMPK activation through Calcium/calmodulin-dependent protein kinase 2 (CaMKK2) and induces autophagy. In addition, AMPK itself increases autophagy by downregulating the mammalian target of rapamycin complex (mTORC). However, whether increased autophagy inhibits or promotes cell death and drug resistance is contradictory. This study reveals that there are numerous pathways other than cell metabolism by which AMPK exerts its effects in the advanced stages of PCa, making it a priceless treatment target. Finally, we mention some drugs developed to treat the advanced stages of PCa by acting on AMPK.

Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin.

BACKGROUND: Acute kidney injury (AKI) is associated with a severe decline in kidney function caused by abnormalities within the podocytes' glomerular matrix. Recently, AKI has been linked to alterations in glycolysis and the activity of glycolytic enzymes, including pyruvate kinase M2 (PKM2). However, the contribution of this enzyme to AKI remains largely unexplored. METHODS: Cre-loxP technology was used to examine the effects of PKM2 specific deletion in podocytes on the activation status of key signaling pathways involved in the pathophysiology of AKI by lipopolysaccharides (LPS). In addition, we used lentiviral shRNA to generate murine podocytes deficient in PKM2 and investigated the molecular mechanisms mediating PKM2 actions in vitro. RESULTS: Specific PKM2 deletion in podocytes ameliorated LPS-induced protein excretion and alleviated LPS-induced alterations in blood urea nitrogen and serum albumin levels. In addition, PKM2 deletion in podocytes alleviated LPS-induced structural and morphological alterations to the tubules and to the brush borders. At the molecular level, PKM2 deficiency in podocytes suppressed LPS-induced inflammation and apoptosis. In vitro, PKM2 knockdown in murine podocytes diminished LPS-induced apoptosis. These effects were concomitant with a reduction in LPS-induced activation of ß-catenin and the loss of Wilms' Tumor 1 (WT1) and nephrin. Notably, the overexpression of a constitutively active mutant of ß-catenin abolished the protective effect of PKM2 knockdown. Conversely, PKM2 knockdown cells reconstituted with the phosphotyrosine binding-deficient PKM2 mutant (K433E) recapitulated the effect of PKM2 depletion on LPS-induced apoptosis, ß-catenin activation, and reduction in WT1 expression. CONCLUSIONS: Taken together, our data demonstrates that PKM2 plays a key role in podocyte injury and suggests that targetting PKM2 in podocytes could serve as a promising therapeutic strategy for AKI. TRIAL REGISTRATION: Not applicable. Video abstract.

Mogroside V Improves Follicular Development and Ovulation in Young-Adult PCOS Rats Induced by Letrozole and High-Fat Diet Through Promoting Glycolysis.

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovaries. In this study, we induced a young-adult PCOS rat model by oral administration of letrozole combined with a high-fat diet and then treated with mogroside V (MV) to evaluate the protective effects of MV on endocrine and follicle development in young-adult PCOS rats. MV (600 mg/kg/day) administration not only significantly reduced the body weight and ovary weight, but also attenuated the disrupted estrous cycle and decreased the level of testosterone. MV restored the follicular development, especially by increasing the number of corpus luteum and the thickness of the granular layer in young-adult POCS rats. Moreover, metabolomics showed that MV markedly increased the levels of D-Glucose 6-phosphate, lactate and GTP, while decreased the level of pyruvate. Bioinformatic analysis revealed that MV recovered multiple metabolism-related processes including gluconeogenesis, glycolysis and glucose metabolic process. Further real-time quantitative PCR analysis showed that MV upregulated the expression of lactate dehydrogenase A (Ldha), hexokinase 2 (Hk2) and pyruvate kinase M2 (Pkm2). Western blotting and immunohistochemistry analysis showed that MV restored the expression of lactate dehydrogenase A (Ldha), hexokinase 2 (Hk2) and pyruvate kinase M2 (Pkm2). Collectively, these findings indicated that MV could effectively improve the ovarian microenvironment by upregulating the expression of LDHA, HK2 and PKM2 in granulosa cells and enhancing lactate and energy production, which may contribute to follicle development and ovulation of young-adult PCOS rats.

PKM2 Is Overexpressed in Glioma Tissues, and Its Inhibition Highly Increases Late Apoptosis in U87MG Cells With Low-density Specificity.

BACKGROUND/AIM: Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme in aerobic glycolysis and is involved in tumor initiation and progression. However, there are few studies on the correlation between PKM2 expression and its role in glioma. MATERIALS AND METHODS: PKM2 expression was immunohistochemically examined in human brain tumor samples. Furthermore, we studied the effects of two PKM2 inhibitors (shikonin and compound 3K) on the U87MG glioma cell line. RESULTS: PKM2 was overexpressed in most glioma tissues when compared to controls. Interestingly, glioma-adjacent tissues from showed slight PKM2 overexpression. This suggests that PKM2 overexpression maybe an important trigger factor for glioma tumorigenesis. We found that the PKM2 inhibitor shikonin was effective against U87MG cells at a relatively low dose and was largely dependent on low cellular density compared to the effects of the anticancer drug vincristine. Shikonin highly increased late-apoptosis of U87MG cells. We also demonstrated that autophagy was involved in the increase in late-apoptosis levels caused by shikonin. Although vincristine treatment led to a high level of G2-phase arrest in U87MG cells, shikonin did not increase G2 arrest. Co-treatment with two PKM2 inhibitors, shikonin and compound 3K, increased the inhibitory effects. CONCLUSION: Combination therapy with PKM2 inhibitors together might be more effective than combination therapy with anticancer drugs. Our findings encourage the application of PKM2-targeting in gliomas, and lay the foundation for the development of PKM2 inhibitors as promising antitumor agents for glioma.

Etavopivat, a Pyruvate Kinase Activator in Red Blood Cells, for the Treatment of Sickle Cell Disease.

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia.

miR-142-3p Modulates Cell Invasion and Migration via PKM2-Mediated Aerobic Glycolysis in Colorectal Cancer.

Decreased expression of miR-142-3p was observed in human cancers. However, the function and mechanism of miR-142-3p in human colorectal cancer remain obscure. The expressions of miR-142-3p in human colorectal cancer tissues and cell lines were measured by RT-qPCR. The effects of miR-142-3p on cell invasion and migration were detected by transwell assays. The efficiency of aerobic glycolysis was determined by glucose consumption and lactate production. Dual-luciferase reporter assays were performed to confirm the correlation between miR-142-3p and pyruvate kinase isozyme M2 (PKM2). The level of PKM2 was assessed by western blotting. Our results showed that the expression of miR-142-3p was decreased both in human colorectal cancer tissues and in cells. Overexpression of miR-142-3p in cell line attenuated colorectal cancer cell invasion and migration. About the underlying mechanism, we found that miR-142-3p modulated aerobic glycolysis via targeting pyruvate kinase M2 (PKM2). In addition, we demonstrated PKM2 and PKM2-mediated aerobic glycolysis contributes to miR-142-3p-mediated colorectal cancer cell invasion and migration. Hence, these data suggested that miR-142-3p was a potential therapeutic target for the treatment of human colorectal cancer.

PKM2 Activator TEPP-46 Attenuates Thoracic Aortic Aneurysm and Dissection by Inhibiting NLRP3 Inflammasome-Mediated IL-1ß Secretion.

BACKGROUND: The development of thoracic aortic aneurysm and dissection (TAAD) is mediated by inflammasome activation, which exacerbates the secretion of pro-inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs), and reactive oxygen species (ROS). The glycolytic enzyme pyruvate kinase M2 (PKM2) has shown a protective role against various disorders with an inflammatory basis, such as sepsis, tumorigenesis, and diabetic nephropathy. However, its potential role in TAAD has not been investigated so far. APPROACH AND RESULTS: We analyzed aortic tissues from TAAD patients and the ß-aminopropionitrile fumarate (BAPN)-induced mouse model of TAAD and observed elevated levels of PKM2 in the aortic lesions of both. Treatment with the PKM2 activator TEPP-46 markedly attenuated the progression of TAAD in the mouse model as demonstrated by decreased morbidity and luminal diameter of the aorta. In addition, the thoracic aortas of the BAPN-induced mice showed reduced monocytes and macrophages infiltration and lower levels of IL-1ß, MMPs, and ROS when treated with TEPP-46. Furthermore, TEPP-46 treatment also suppressed the activation of the NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome by downregulating p-STAT3 and HIF1-α. CONCLUSION: Pyruvate kinase M2 plays a protective role in TAAD development, and its activation is a promising therapeutic strategy against the progression of TAAD.

Pyruvate Kinase M2 Increases Angiogenesis, Neurogenesis, and Functional Recovery Mediated by Upregulation of STAT3 and Focal Adhesion Kinase Activities After Ischemic Stroke in Adult Mice.

Ischemic stroke remains a serious threat to human life. Generation of neuronal and vascular cells is an endogenous regenerative mechanism in the adult brain, which may contribute to tissue repair after stroke. However, the regenerative activity is typically insufficient for significant therapeutic effects after brain injuries. Pyruvate kinase isoform M2 (PKM2) is a key regulator for energy metabolism. PKM2 also has nonmetabolic roles involving regulations of gene expression, cell proliferation, and migration in cancer cells as well as noncancerous cells. In a focal ischemic stroke mouse model, recombinant PKM2 (rPKM2) administration (160 ng/kg, intranasal delivery) at 1 h after stroke showed the significant effect of a reduced infarct volume of more the 60%. Delayed treatment of rPKM2, however, lost the acute neuroprotective effect. We then tested a novel hypothesis that delayed treatment of PKM2 might show proregenerative effects for long-term functional recovery and this chronic action could be mediated by its downstream STAT3 signaling. rPKM2 (160 ng/kg) was delivered to the brain using noninvasive intranasal administration 24 h after the stroke and repeated every other day. Western blot analysis revealed that, 7 days after the stroke, the levels of PKM2 and phosphorylated STAT3 and the expression of angiogenic factors VEGF, Ang-1, and Tie-2 in the peri-infarct region were significantly increased in the rPKM2 treatment group compared with those of the stroke vehicle group. To label proliferating cells, 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg, i.p.) was injected every day starting 3 days after stroke. At 14 days after stroke, immunohistochemistry showed that rPKM2 increased cell homing of doublecortin (DCX)-positive neuroblasts to the ischemic cortex. In neural progenitor cell (NPC) cultures, rPKM2 (0.4-4 nM) increased the expression of integrin ß1 and the activation/phosphorylation of focal adhesion kinase (FAK). A mediator role of FAK in PKM2-promoted cell migration was verified in FAK-knockout fibroblast cultures. In the peri-infarct region of the brain, increased numbers of Glut-1/BrdU and NeuN/BrdU double-positive cells indicated enhanced angiogenesis and neurogenesis, respectively, compared to stroke vehicle mice. Using Laser Doppler imaging, we observed better recovery of the local blood flow in the peri-infarct region of rPKM2-treated mice 14 days after stroke. Meanwhile, rPKM2 improved the sensorimotor functional recovery measured by the adhesive removal test. Inhibiting the STAT3 phosphorylation/activation by the STAT3 inhibitor, BP-1-102 (3 mg/kg/day, o.g.), abolished all beneficial effects of rPKM2 in the stroke mice. Taken together, this investigation provides the first evidence demonstrating that early treatment of rPKM2 shows an acute neuroprotective effect against ischemic brain damage, whereas delayed rPKM2 treatment promotes regenerative activities in the poststroke brain leading to better functional recovery. The underlying mechanism involves activation of the STAT3 and FAK signals in the poststroke brain.

A Fluorescent, Reagentless Biosensor for ATP, Based on Malonyl-Coenzyme A Synthetase.

A fluorescent reagentless biosensor for ATP has been developed, based on malonyl-coenzyme A synthetase from Rhodopseudomonas palustris as the protein scaffold and recognition element. Two 5-iodoacetamidotetramethylrhodamines were covalently bound to this protein to provide the readout. This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It measures ATP concentrations with micromolar sensitivity and is highly selective for ATP relative to ADP. Its ability to monitor enzymatic ATP production or depletion was demonstrated in steady-state kinetic assays in which ATP is a product or substrate, respectively.

Diffusion restrictions surrounding mitochondria: a mathematical model of heart muscle fibers.

Several experiments on permeabilized heart muscle fibers suggest the existence of diffusion restrictions grouping mitochondria and surrounding ATPases. The specific causes of these restrictions are not known, but intracellular structures are speculated to act as diffusion barriers. In this work, we assume that diffusion restrictions are induced by sarcoplasmic reticulum (SR), cytoskeleton proteins localized near SR, and crowding of cytosolic proteins. The aim of this work was to test whether such localization of diffusion restrictions would be consistent with the available experimental data and evaluate the extent of the restrictions. For that, a three-dimensional finite-element model was composed with the geometry based on mitochondrial and SR structural organization. Diffusion restrictions induced by SR and cytoskeleton proteins were varied with other model parameters to fit the set of experimental data obtained on permeabilized rat heart muscle fibers. There are many sets of model parameters that were able to reproduce all experiments considered in this work. However, in all the sets, <5-6% of the surface formed by SR and associated cytoskeleton proteins is permeable to metabolites. Such a low level of permeability indicates that the proteins should play a dominant part in formation of the diffusion restrictions.

[Effects of plasminogen, streptokinase and their equimolar complexes with pyruvate kinase on the human neuroblastoma IMR-32 cells].

The system of extracellular proteolysing, consists of plasminogen (PGn), its active protease (plasmin), PGn activation and PGn activators inhibitors, influences the nervous tissue functions, their growth, differentiation and proliferation in both, normal and pathological conditions. The purpose of the investigation was to study the effects of exogenous PGn, its activator streptokinase (SK), PK and their equimolar complex on the morpho-functional state neuroblastoma IMR-32 cells. PGn, SK, PK and their complexes stimulated cells proliferation during 1-3 days of incubation, shown by cell quantity increase. We also observed DNA, RNA and protein increase. The low lactate dehydrogenase efflux was evidence of that an addition of the proteins under investigation in the culture medium prevented the development of degenerative alterations connected with serum deprivation. The levels of extracellular PGn-activator activity, as measured by the biochemical fibrinolytic assay, increased over SK. This SK effect vanished on the 3rd day when SK formed complexes with PK. New original facts obtained testify the probability of initiation of neoplastic transformation and tumor growth potentiation.

Na+-K+ pumps in the transverse tubular system of skeletal muscle fibers preferentially use ATP from glycolysis.

The Na(+)-K(+) pumps in the transverse tubular (T) system of a muscle fiber play a vital role keeping K(+) concentration in the T-system sufficiently low during activity to prevent chronic depolarization and consequent loss of excitability. These Na(+)-K(+) pumps are located in the triad junction, the key transduction zone controlling excitation-contraction (EC) coupling, a region rich in glycolytic enzymes and likely having high localized ATP usage and limited substrate diffusion. This study examined whether Na(+)-K(+) pump function is dependent on ATP derived via the glycolytic pathway locally within the triad region. Single fibers from rat fast-twitch muscle were mechanically skinned, sealing off the T-system but retaining normal EC coupling. Intracellular composition was set by the bathing solution and action potentials (APs) triggered in the T-system, eliciting intracellular Ca(2+) release and twitch and tetanic force responses. Conditions were selected such that increased Na(+)-K(+) pump function could be detected from the consequent increase in T-system polarization and resultant faster rate of AP repriming. Na(+)-K(+) pump function was not adequately supported by maintaining cytoplasmic ATP concentration at its normal resting level ( approximately 8 mM), even with 10 or 40 mM creatine phosphate present. Addition of as little as 1 mM phospho(enol)pyruvate resulted in a marked increase in Na(+)-K(+) pump function, supported by endogenous pyruvate kinase bound within the triad. These results demonstrate that the triad junction is a highly restricted microenvironment, where glycolytic resynthesis of ATP is critical to meet the high demand of the Na(+)-K(+) pump and maintain muscle excitability.

In vitro and in vivo expression of human erythrocyte pyruvate kinase in erythroid cells: a gene therapy approach.

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

Interdependent effects of inorganic phosphate and creatine phosphate on sarcoplasmic reticulum Ca2+ regulation in mechanically skinned rat skeletal muscle.

1. The effects of creatine phosphate (CP) and inorganic phosphate (Pi) on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in mechanically skinned muscle fibres from rat extensor digitorum longus (EDL) muscles. Changes in [Ca2+] were detected using fura-2 fluorescence, during continuous perfusion or when the solution surrounding the preparation was restricted to approximately 6 microl by stopping perfusion. 2. In solutions with 5 mM ATP and 10 mM CP, stopping the flow for 2-3 min had no effect on [Ca2+] within the bath. This suggests that SR Ca2+ uptake is balanced by an efflux under these conditions. 3. In solutions with CP, the introduction of Pi induced a small transient rise in [Ca2+], due to Ca2+ loss from the SR. Following equilibration with solutions containing Pi (> or = 5 mM), a maintained decrease in [Ca2+] occurred when the flow was stopped. This is consistent with calcium phosphate (Ca-Pi) precipitation within the SR, resulting in maintained Ca2+ uptake. 4. In the absence of CP, the [Ca2+] within the bath increased progressively when the flow was stopped. This rise in [Ca2+] was inhibited by an alternative ATP regenerating system comprising phosphoenolpyruvate (PEP) and pyruvate kinase (PK). Therefore, the loss of Ca2+ from the SR may result from local ADP accumulation and the consequent reversal of the SR Ca2+ pump. 5. In the absence of CP, the initial Ca2+ release associated with the introduction of Pi increased markedly. Following prolonged equilibration with solutions containing Pi, a rise in [Ca2+] occurred within the bath when the flow was stopped. Maintained Ca2+ uptake associated with Ca-Pi precipitation was not apparent at any level of Pi tested (1-60 mM), when CP was absent. 6. These results suggest that withdrawal of CP is associated with activation of a SR Ca2+ efflux pathway. This may involve reversal of the SR Ca2+ pump, due to local ADP accumulation. In the absence of CP, the dominant influence of Pi appears to involve further Ca2+ efflux via the SR Ca2+ pump. The possible relevance of these effects to skeletal muscle fatigue is considered.