Inhibition of free radical initiated peroxidation of human erythrocyte ghosts by flavonols and their glycosides

The in vitro peroxidation of human erythrocyte ghosts was used as a model to study the free radical-induced damage of biological membranes and the protective effect of flavonols and their glycosides, i.e., quercetin (Q), quercetin galactopyranoside (QG), quercetin rhamnopyranoside (QR), rutin (R), morin (MO), kaempferol (K) and kaempferol glucoside (KG). The peroxidation was initiated by a water-soluble free radical initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AMPAD) at physiological temperature, and monitored by oxygen uptake. Kinetic analysis of the peroxidation process demonstrates that these flavonols and their glycosides are effective antioxidants against AMPAD-initiated oxidative damage of human erythrocyte ghosts, and that the flavonols bearing ortho-dihydroxyl groups possess significantly higher antioxidant activity than those bearing no such functionalities and the glycosides are less active than their parent aglycones.     

Effect of Hydrolyzable Tannins on Glucose-Transporter Expression and Their Bioavailability in Pig Small-Intestinal 3D Cell Model

Intestinal transepithelial transport of glucose is mediated by glucose transporters, and affects postprandial blood-glucose levels. This study investigates the effect of wood extracts rich in hydrolyzable tannins (HTs) that originated from sweet chestnut (Castanea sativa Mill.) and oak (Quercus petraea) on the expression of glucose transporter genes and the uptake of glucose and HT constituents in a 3D porcine-small-intestine epithelial-cell model. The viability of epithelial cells CLAB and PSI exposed to different HTs was determined using alamarBlue^®. qPCR was used to analyze the gene expression of SGLT1, GLUT2, GLUT4, and POLR2A. Glucose uptake was confirmed by assay, and LC–MS/ MS was used for the analysis of HT bioavailability. HTs at 37 µg/mL were found to adversely affect cell viability and downregulate POLR2A expression. HT from wood extract Tanex at concentrations of 4 µg/mL upregulated the expression of GLUT2, as well as glucose uptake at 1 µg/mL. The time-dependent passage of gallic acid through enterocytes was influenced by all wood extracts compared to gallic acid itself as a control. These results suggest that HTs could modulate glucose uptake and gallic acid passage in the 3D cell model.     

Study on glucose transport in muscle cells by extracts from Mitragyna speciosa (Korth) and mitragynine

The leaves of Mitragyna speciosa Korth (Rubiaceae) have been used in folk medicine for its unique medicinal properties. This study examined the water, methanolic and crude alkaloidal extracts from M. speciosa leaves and its major constituent mitragynine for the enhancement of glucose transport. Cellular uptake of radioactive 2-deoxyglucose was determined in rat L8 myotubes. Involving signalling pathway was determined with the specific inhibitors. Cell cytotoxicity was monitored by lactate dehydrogenase assay. Protein levels of glucose transporters (GLUTs) were measured by Western blotting. The results show that test samples significantly increased the rate of glucose uptake. The uptake was associated with increase in GLUT1 protein content. Co-incubation with insulin had no additional effect, but the cellular uptake was decreased by wortmannin and SB 203580, specific inhibitors of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK), respectively. It is concluded that the increased glucose transport activity of M. speciosa is associated with increases in activities of the key enzymes dependent to the insulin-stimulated glucose transport for its acute action, and increases in the GLUT1 content for its long-term effect. This study demonstrated the effect of M. speciosa in stimulating glucose transport in muscle cells, implicating the folkloric use of M. speciosa leaves for treating diabetes.     

BILIRUBIN-PHOTOSENSITIZED LYSIS OF RESEALED ERYTHROCYTE MEMBRANES*

Abstract— The ability of bilirubin to photosensitize biomembrane damage has been tested, using resealed human erythrocyte ghosts as a model system. Continuous irradiation of bilirubin-charged ghosts with broad-band blue light resulted in membrane lysis, as evidenced by the release of trapped markers such as Na⁺ or glucose-6-phosphate. Sodium dodecyl sulfate-gel electrophoresis of membrane proteins revealed a progressive and relatively rapid loss of spectrin (bands 1 and 2) and band 4.1 during irradiation, accompanied by the appearance of cross-linked polypeptides. The antioxidant butylated hydroxytoluene protected ghosts against lysis, but not against protein cross-linking, suggesting that lipid peroxidation is the crucial membrane disrupting event.     

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices

The observation that ascorbate known to retain pro-oxidant properties induces cell death in a number of immortal cell lines, led us to examine its mechanism and whether it is involved in oxidative stress injury in such asocorbate-enriched tissue cells as hepatocytes. In rat liver homogenates, higher concentrations (1 and 3 mM) of ascorbate suppressed lipid peroxide productions but lower concentrations (0.1 and 0.3 mM) did not. In contrast to the homogenate, ascorbate increased lipid peroxide production in liver slices in a concentration dependant manner. Iso-ascorbate, the epimer of ascorbate did not cause an increase the oxidative stress in liver slices. This differential effect between homogenates and liver slices implies that cellular integrity is required for ascorbate to induce oxidative stress. Wortmannin, an inhibitor of the GLUT (glucose transporter) thought to transport dehydroascorbate into cells, inhibited [(14)C]-ascorbate uptake and suppressed oxidative stress in liver slices. Wortmannin suppressed that [(14)C]-ascorbate uptake by GLUT following oxidation to [(14)C]dehydroascorbate. Taken together, these observations support our hypothesis that ascorbate is oxidized to dehydroascorbate by molecular oxygen in solution (i.e., plasma and culture medium) which is then carried into hepatocytes (via a GLUT) where it is reduced back to ascorbate causing oxidative stress.     

Synthesis of Indomorphan Pseudo‐Natural Product Inhibitors of Glucose Transporters GLUT‐1 and ‐3

Bioactive compound design based on natural product (NP) structure may be limited because of partial coverage of NP‐like chemical space and biological target space. These limitations can be overcome by combining NP‐centered strategies with fragment‐based compound design through combination of NP‐derived fragments to afford structurally unprecedented “pseudo‐natural products” (pseudo‐NPs). The design, synthesis, and biological evaluation of a collection of indomorphan pseudo‐NPs that combine biosynthetically unrelated indole‐ and morphan‐alkaloid fragments are described. Indomorphane derivative Glupin was identified as a potent inhibitor of glucose uptake by selectively targeting and upregulating glucose transporters GLUT‐1 and GLUT‐3. Glupin suppresses glycolysis, reduces the levels of glucose‐derived metabolites, and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT‐1 and GLUT‐3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity.     

Gundelia tournefortii: Fractionation,Chemical Composition and GLUT4 Translocation Enhancement in Muscle Cell Line

Type 2 diabetes (T2D) is a chronic metabolic disease, which could affect the daily life of patients and increase their risk of developing other diseases. Synthetic anti-diabetic drugs usually show severe side effects. In the last few decades, plant-derived drugs have been intensively studied, particularly because of a rapid development of the instruments used in analytical chemistry. We tested the efficacy of Gundelia tournefortii L. (GT) in increasing the translocation of glucose transporter-4 (GLUT4) to the myocyte plasma membrane (PM), as a main strategy to manage T2D. In this study, GT methanol extract was sub-fractionated into 10 samples using flash chromatography. The toxicity of the fractions on L6 muscle cells, stably expressing GLUTmyc, was evaluated using the MTT assay. The efficacy with which GLUT4 was attached to the L6 PM was evaluated at non-toxic concentrations. Fraction 6 was the most effective, as it stimulated GLUT4 translocation in the absence and presence of insulin, 3.5 and 5.2 times (at 250 μg/mL), respectively. Fraction 1 and 3 showed no significant effects on GLUT4 translocation, while other fractions increased GLUT4 translocation up to 2.0 times. Gas chromatography–mass spectrometry of silylated fractions revealed 98 distinct compounds. Among those compounds, 25 were considered anti-diabetic and glucose disposal agents. These findings suggest that GT methanol sub-fractions exert an anti-diabetic effect by modulating GLUT4 translocation in L6 muscle cells, and indicate the potential of GT extracts as novel therapeutic agents for T2D.     

β-Sitosterol-D-Glucopyranoside Mimics Estrogenic Properties and Stimulates Glucose Utilization in Skeletal Muscle Cells

Estrogenic molecules have been reported to regulate glucose homeostasis and may be beneficial for diabetes management. Here, we investigated the estrogenic effect of β-sitosterol-3-O-D-glucopyranoside (BSD), isolated from the fruits of Cupressus sempervirens and monitored its ability to regulate glucose utilization in skeletal muscle cells. BSD stimulated ERE-mediated luciferase activity in both ERα and ERβ-ERE luc expression system with greater response through ERβ in HEK-293T cells, and induced the expression of estrogen-regulated genes in estrogen responsive MCF-7 cells. In silico docking and molecular interaction studies revealed the affinity and interaction of BSD with ERβ through hydrophobic interaction and hydrogen bond pairing. Furthermore, prolonged exposure of L6-GLUT4myc myotubes to BSD raised the glucose uptake under basal conditions without affecting the insulin-stimulated glucose uptake, the effect associated with enhanced translocation of GLUT4 to the cell periphery. The BSD-mediated biological response to increase GLUT4 translocation was obliterated by PI-3-K inhibitor wortmannin, and BSD significantly increased the phosphorylation of AKT (Ser-473). Moreover, BSD-induced GLUT4 translocation was prevented in the presence of fulvestrant. Our findings reveal the estrogenic activity of BSD to stimulate glucose utilization in skeletal muscle cells via PI-3K/AKT-dependent mechanism.     

绿茶多酚对自由基诱导的红细胞膜过氧化的抑制作用

采用水溶性偶氮引发剂2,2'-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)在37引发入血红细胞膜的过氧化,通过测定氧气吸收及维生素E的消耗研究了过氧化过程的动力学,并对从绿茶中提取的主要多酚类化合物的抗氧化活性做了定量研究。使用的绿茶多酚有:(-)-表儿茶素(EC),(-)-表儿茶素(EGC),(-)-表儿茶素酸酯(ECG)和(-)-表儿茶素培酸酯(EGCG)。结果表明,这些绿茶多酚能够显著缩短过氧化反应的动力学链长,有效地抑制红细胞膜的过氧化。抗氧化活性顺序为:EC〉GCG〉EGCG〉EGC。     

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC₅₀ value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP‐dependent kinase, inhibition of mTOR signaling, and induction of cell‐cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.     

Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.     

Stevioside Attenuates Insulin Resistance in Skeletal Muscle by Facilitating IR/IRS-1/Akt/GLUT 4 Signaling Pathways: An In Vivo and In Silico Approach

Type-2 diabetes mellitus (T2DM), the leading global health burden of this century majorly develops due to obesity and hyperglycemia-induced oxidative stress in skeletal muscles. Hence, developing novel drugs that ameliorate these pathological events is an immediate priority. The study was designed to analyze the possible role of Stevioside, a characteristic sugar from leaves of Stevia rebaudiana (Bertoni) on insulin signaling molecules in gastrocnemius muscle of obesity and hyperglycemia-induced T2DM rats. Adult male Wistar rats rendered diabetic by administration of high fat diet (HFD) and sucrose for 60 days were orally administered with SIT (20 mg/kg/day) for 45 days. Various parameters were estimated including fasting blood glucose (FBG), serum lipid profile, oxidative stress markers, antioxidant enzymes and expression of insulin signaling molecules in diabetic gastrocnemius muscle. Stevioside treatment improved glucose and insulin tolerances in diabetic rats and restored their elevated levels of FBG, serum insulin and lipid profile to normalcy. In diabetic gastrocnemius muscles, Setvioside normalized the altered levels of lipid peroxidase (LPO), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH*), antioxidant enzymes (CAT, SOD, GPx and GSH) and molecules of insulin signaling including insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt mRNA levels. Furthermore, Stevioside enhanced glucose uptake (GU) and oxidation in diabetic muscles by augmenting glucose transporter 4 (GLUT 4) synthesis very effectively in a similar way to metformin. Results of molecular docking analysis evidenced the higher binding affinity with IRS-1 and GLUT 4. Stevioside effectively inhibits oxidative stress and promotes glucose uptake in diabetic gastrocnemius muscles by activating IR/IRS-1/Akt/GLUT 4 pathway. The results of the in silico investigation matched those of the in vivo study. Hence, Stevioside could be considered as a promising phytomedicine to treat T2DM.     

Oxygen sensitivity of red cell membrane transporters revisited

In this paper, we provide an update on O2-dependent membrane transport in red cells. O2-sensitive membrane transport was compared in nucleated (chicken) and enucleated (human) red cells, to investigate effects on organic (glucose transporter [GLUT]) and inorganic (K(+)-Cl- cotransporter [KCC]/Na(+)-K(+)-2Cl- cotransporter [NKCC]) transporters, to study the response of so-called "housekeeping" transporters (Na+/K+ pump and anion exchanger [AE]) and, finally, to compare O2 sensitivity in normal human red cells with those from sickle cell patients. The Na+/K+ pump showed no change in activity between oxygenated and deoxygenated cells in any of the samples. KCC in normal human red cells had the greatest O2 sensitivity, being stimulated some 20-fold on oxygenation. It was more modestly stimulated by O2 in chicken red cells and HbS cells. By contrast, NKCC was stimulated by deoxygenation in all cases. GLUT showed little response to O2 tension, other than a small stimulation in deoxygenated chicken red cells. Finally, AE1 was stimulated by oxygenation in HbA cells, but this stimulation by O2 was absent in HbS cells and pink ghosts prepared from HbA cells. The significance of these findings is discussed.     

缬氨霉素液膜传输Rb＋跨人红细胞膜行为的研究

通过石墨炉原子吸收法破膜检测胞内Rb＋的含量, 考察大环配体缬氨霉素运载Rb＋跨膜的行为. 基于传输过程的动力学研究, 讨论了离子跨细胞膜时影响离子传输速率和跨膜量的各种因素. 同时还以荧光法研究了RbCl对红细胞膜蛋白荧光强度的作用. 结果显示, 缬氨霉素传输Rb＋跨人红细胞膜的行为呈现串联一级反应动力学特征, 跨膜量主要受配体浓度、温育时间的影响. 高浓度RbCl能够影响膜蛋白的荧光强度.     

Photodynamic inactivation of retroviruses by phthalocyanines: the effects of sulphonation, metal ligand and fluoride.

The photodynamic inactivation of retroviruses was investigated using aluminium and zinc phthalocyanine (Pc) derivatives. The N2 retrovirus packaged in either of the two murine cell lines, Psi2 and PA317, was used as a model for enveloped viruses. AlPc derivatives were found to be more effective photodynamically for inactivation of the viruses than the corresponding ZnPc derivatives. Sulphonation of the Pc macrocycle reduced its photodynamic activity progressively for both AlPc and ZnPc. Fluoride at 5 mM during light exposure completely protected viruses against inactivation by AlPc. In the presence of F-, inactivation by the sulphonated derivatives AlPcS1 and AlPcS4 was reduced 2.5- and twofold respectively. In a biological membrane (erythrocyte ghosts), F- had no significant effect on AlPcS4-sensitized lipid peroxidation. Under similar conditions, cross-linking of spectrin monomers in ghosts is drastically inhibited (E. Ben-Hur and A. Orenstein, Int. J. Radiat. Biol., 60 (1991) 293-301). Since Pc derivatives do not inactivate non-enveloped viruses, it is hypothesized that inactivation occurs by photodynamic damage to envelope protein(s). Substitution of sulphonic acid residues reduces the binding of Pc derivatives to the envelope protein(s), thereby diminishing their photodynamic efficacy and the ability of F- to modify it.     

Interaction of pentoxifylline with human erythrocytes. I. Interaction of xanthine derivatives with human erythrocyte ghosts

The interaction of pentoxifylline and other xanthine derivatives with human erythrocyte ghosts was studied. By fluorescence spectroscopy it was found that xanthine derivatives have two modes of binding to erythrocyte ghosts. One is a high-capacity binding to erythrocyte membranes. It seems that the 5-oxohexyl side chain of pentoxifylline is important for this. The second type may be a binding to proteins on the membranes and is specific for pentoxifylline and caffeine. From the circular dichroism spectra, it was presumed that the second binding mode of pentoxifylline occurs at hydrophobic regions of beta-structure of the membrane proteins. The relative high specificity in the interaction of pentoxifylline with erythrocytes should be related to its unique physiological activity on erythrocytes.     

Isolation of Hemoglobin from Bovine Erythrocytes by Controlled Hemolysis in the Membrane Bioreactor

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2–7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.     

Two new compounds of Penicillium polonicum,an endophytic fungus from Camptotheca acuminata Decne

Abstract

To discover novel structural compounds which are producted by endophytic fungi, a primary chemical profiling of Camptotheca acuminata Decne derived endophytic fungus Penicillum polonicum had been taken. Two new compounds β-lactone polonicin A (1) and enoic acid polonicin B (2) together with seven known compounds 3-9 were isolated from Penicillum polonicum obtained from C. acuminata. The structures of the new compounds 1 and 2 were identified by modern spectrum technology including detailed 1D, 2D NMR and MS data analyses. When tested against HepG2 hepatocellular carcinoma (HCC) cell lines, compounds 4-8 showed moderate anti-HCC activity. In addition, compound 1-3 have effects on increasing GLUT4 translocation and glucose uptake in vitro. Compound 1 showed the strongest glucose uptake and GLUT4 translocation activities in rat skeleton (L6) myoblast cell line with enhancements of 1.8 and 2.1 folds respectively compared to the control.