Implication of phosphorylation of the myosin II regulatory light chain in insulin-stimulated GLUT4 translocation in 3T3-F442A adipocytes

In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca(2+)/calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca(2+) in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3-F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca(2+)-dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin-stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca(2+)/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes.     

Formation of 3-phenylhydrazono-3-aroylpropionamides from the reaction of 5-aroyl-3(2H)-isothiazolones with phenylhydrazine

Reaction of 2-substituted-5-aroyl-3(2H)-isothiazolones 1 with phenylhydrazine in refluxing ethanol was found to give sulfur-free products, which were characterized as 3-phenylhydrazono-3-aroyl-N-substituted-pro-pionamides 4. Acid hydrolysis of the 3-benzoyl compounds 4 was shown to yield 1-phenyl-3-benzoyl-2-pyrazolin-5-one ( 7 ), whose structure was confirmed by an independent synthesis starting with the chloride of 1-phenyl-5-methoxypyrazole-3-carboxylic acid ( 10 ).     

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.     

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.     

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.     

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.     

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.     

Discovery of New Glucose Uptake Inhibitors as Potential Anticancer Agents by Non-Radioactive Cell-Based Assays

Tumor cells rely on aerobic glycolysis to support growth and survival, thus require more glucose supply. Glucose transporters GLUTs, primarily GLUT1, are overexpressed in various cancers. Targeting GLUTs has been regarded as a promising anticancer strategy. In this study, we first evaluated 75 potential GLUT1 inhibitors obtained from virtual screening of the NCI chemical library by a high-throughput cell-based method using a fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-d-glucose (2-NBDG) in COS-7 and SKOV3 cells that express high levels of GLUT1. Four compounds, #12, #16, #43 and #69, that significantly inhibited glucose uptake were further evaluated using flow cytometry directly measuring 2-NBDG uptake at the single-cell level and a Glucose Uptake-Glo^TM assay indirectly measuring 2-deoxy-d-glucose uptake in SKOV3, COS-7 or MCF-7 cells. The inhibitory effect on cancer cell growth was also determined in SKOV3 and MCF-7 cells, and #12 exhibited the best growth inhibitory effect equivalent to a known GLUT1 inhibitor WZB117. Although the anticancer effect of the identified potential GLUT1 inhibitors was moderate, they may enhance the activity of other anticancer drugs. Indeed, we found that #12 synergistically enhanced the anticancer activity of metformin in SKOV3 ovarian cancer cells.     

The solid-state michael addition of indole to 4-arylidene-3-methyl-5-pyrazolone

The Michael addition reactions of indole to 4-arylidene-3-methyl-1-phenyl-5-pyrazolone 1 were investigated in the solid state, by which a series of new compounds, 1-aryl-1-(3-indolyl)-1-(3′-methyl-1′-phenyl-5′-pyrazolon-4′-yl)methanes 2 , were easily obtained. This provides a feasible method of preparing novel compounds containing two heterocyclic groups at the same carbon.     

3-甲基-5-苯基-2-芳基吗啉盐酸盐合成及其抗实验性抑郁活性

苯基氨基乙醇;芳基吗啉;合成;抗抑郁活性     

Pyridazines. Part 31: synthesis and antiplatelet activity of 4,5-disubstituted-6-phenyl-3(2H)-pyridazinones

This paper describes the synthesis and the antiplatelet activity of a series of 4,5-disubstituted-6-phenyl-3(2H)-pyridazinones. Some of these compounds show a dose-dependent activity and were found to be more active than their 5-substituted analogues.     

A convenient synthesis of 3-alkyl-2,3-dihydro-2,4-dioxo-6-phenyl-, 3-alkyl-2,3-dihydro-4-oxo-6-phenyl-2-thioxo-, and 3-alkyl-2-arylimino-2,3-dihydro-4-oxo-6-phenyl-4H-1,3-thiazines

The reactions of 1-alkylamino-1-alkylthio-3-phenylpropene-3-thiones 3 with thiophosgene and phosgene in toluene, followed by treatment of the reaction mixture with triethylamine gave 3-alkyl-2,3-dihydro-4-oxo-6-phenyl-2-thioxo- 4 , 3-alkyl-2,3-dihydro-2,4-dioxo-6-phenyl-4H-1,3-thiazines 5 , respectively in good to excellent yields. Similarly treatment of compounds 3 with N-arylimidoyl dichloride in benzene at room temperature gave 3-alkyl-2-arylimino-2,3-dihydro-4-oxo-6-phenyl-4H-1,3-thiazines 6 in excellent yields. The reactions of compounds 3 with oxalyl chloride in toluene gave also 5 in good yields.     

Studies on the chemistry of 2-(2-oxo-3-phenylpropyl)-benzaldehydes: novel total synthesis of 3-phenylnaphthalen-2-ols and 2-hydroxy-3-phenyl-1,4-naphthoquinones

We describe the first studies on the chemistry of 2-(2-oxo-3-phenylpropyl)benzaldehydes, which were converted into 3-benzylisochromen-1-ones via the corresponding 2-(2-oxo-3-phenylpropyl)benzoic acid. The 2-(2-oxo-3-phenylpropyl)benzaldehydes proved to be convenient starting materials for the synthesis of 3-phenyl-2-naphthols. Oxidation of the latter compounds resulted in a novel, efficient synthesis of 3-phenyl-1,2-naphthoquinones, which were efficiently transformed into 2-hydroxy-3-phenyl-1,4-naphthoquinones.     

(3-苄基-4-芳酰基-1,2,4-三唑-5-基)(5-本基-1,3,4-噁二唑-2-基)硫醚的合成

通过3-苄基-4-芳酰基-1,2,4-三唑-5-巯基负离子在2-甲磺酰基-5-苯基-1,3,4-噁二唑环2-位上的亲核取代反应,制得13个新的(3-苄基-4-芳酰基-1,2,4-三唑-5-基)(5-苯基-1,3,4-噁二唑-2-基)硫醚衍生物.经元素分析、IR、~1H NMR和MS裂解碎片分析确认结构.初步观察了它们在0.01％浓度时对大肠杆菌繁殖的抑制作用.     

葡萄糖转运蛋白4功能调节信号通路及其在药物研发中的应用

葡萄糖转运蛋白4(glucose transporter 4,GLUT4)是胰岛素响应组织骨骼肌和脂肪组织内负责葡萄糖吸收的转运蛋白,它与生物体糖代谢过程密切相关.在肥胖或以胰岛素抵抗为特征的2型糖尿病等代谢性疾病中,GLUT4功能受损;反之,GLUT4功能的变化也能影响整体的糖代谢水平.本文概述了GLUT4的功能、组织分布、功能调节方式以及调控GLUT4功能的小分子化合物的研究进展,讨论了GLUT4在其他疾病中的应用,并展望了其未来研究方向.     

Design, synthesis and hypoglycemic activity of 3-methyl-1-phenyl-4-{4-[(5-methyl-2-phenyloxazol-4-yl)methoxy]benzylene(benzyl)}-2-pyrazol-5-one

Based on the SAR (structure activity relationship) of TZDs (thiazolidinediones), 3-methyl-1-phenyl-2-pyrazoline-5-one was selected as a substitute for TZD. Compounds of 3-methyl-1-phenyl-4-{4-[(5-methyl-2-phenyloxazol-4-yl)methoxy]benzylene(benzyl)}-2-pyrazol-5-one were designed and synthesized to find some more hypoglycemic active agents and further investigate the SAR of this class of compounds. Butanedione monoxime reacted with (substituted) benzaldehyde via cyclization and chlorination to give 4-(chloromethyl)-5-methyl-2-phenyloxazole derivatives, which condensed with 4-hydroxybenzaldehyde or vanillin, and was followed by the Knoevenagel reaction with 3-methyl-1-phenyl-2-pyrazol-5-one to give compounds Ia-Ih. Compounds Ia-Ih were hydrogenated with Pd-C to give IIa-IIh, and their hypoglycemic activity was evaluated with a glucose oxidase kit and insulin load test on normal mice. Sixteen new target compounds were synthesized. All the compounds were characterized by ¹H NMR, IR, MS and elemental analysis. The preliminary pharmacological tests show that the compounds have good hypoglycemic activity and can enhance the action of insulin, especially Ib, Id and If. __________ Translated from Chemical Journal of Chinese Universities, 2007, 28(4): 672–677 [译自: 高等学校化学学报]     

Synthesis and properties of 2-imino-3-benzyl-5-phenyl-1, 3, 4-oxadiazoline

2-Imino-3-benzyl-5-phenyl-1, 3, 4-oxadiazoline (Ia) was synthesized by the reaction of benzyl chloride with 2-amino-5-phenyl-1, 3, 4-oxadiazole (II). A number of imino-group derivatives of Ia were prepared. The structure of the compounds prepared were confirmed by means of their IR spectra.     

Coordination compounds of some 3d metals with 3-methyl-1-phenyl-4-formylpyrazol-5-one

CuL₂ · 1.5H₂O and ML₂ · 2H₂O · 2EtOH (M = Co²⁺, Ni²⁺, Mn²⁺) coordination compounds were synthesized via the exchange reaction between the sodium salt of 3-methyl-1-phenyl-4-formylpyrazol-5-one (HL) and metal chlorides.The synthesized compounds were studied by thermogravimetry, magnetochemistry, and electron and IR spectroscopy. The complexes CuL₂ · 2Py and CoL₂ · 2Py · MeOH were obtained via recrystallization from a methanol-pyridine mixture, and their structures were studied by X-ray diffraction. Pyrazolone was found to be coordinated in the deprotonated enol form and to form six-membered chelate rings with a metal. The coordination polyhedron of a metal cation was found to be a square bipyramid (Cu²⁺) or an axially elongated octahedron (Co²⁺) with its vertices occupied by the oxygen atoms of 3-methyl-1-phenyl-4-formylpyrazol-5-one and the nitrogen atoms of pyridine.     

Mannose/glucose-functionalized dendrimers to investigate the predictable tunability of multivalent interactions

G4-, G5-, and G6-PAMAM dendrimers were functionalized with mixtures of mannose and glucose in varying ratios, and the relative affinities of these compounds for Concanavalin A (Con A) were evaluated using the hemagglutination assay. As the ratio of mannose to glucose increases, the relative activity in the hemagglutination assay (on a per sugar basis) increases linearly. Methyl mannose binds to Con A with an affinity 4-fold higher than that of methyl glucose; multivalency amplifies this trend. The mannose/glucose-functionalized dendrimer results reported here suggest that the affinity of multivalent associations can be attenuated in predictable, reliable ways based on monovalent affinities of the ligands.