Regulation of the expression and function of glucose transporter-1 by TGF-β1 and high glucose in mesangial cells

Objective To study the effects of high glucose and transforming growth factor-β1 (TGF-β1) on the expression and function of glucose transporter-1 (GLUT1) in mouse mesangial cells.Methods Cultured mouse mesangial cells were used.The expression of GLUT1 mRNA was detected by Northern Blot; glucose uptake and its kinetics were determined with a 2-Deoxy-［(3)H］-D-glucose uptake assay.Results Mesangial cells exposed to enriched glucose medium (20 mmol/L) for 72 hours demonstrated a decrease in both GLUT1 mRNA and V(max) for uptake of the glucose analog, 2-deoxy-D-glucose　(2DOG), as compared to mesangial cells cultured in physiologic glucose concentrations(5.5 mmol/L).In contrast, hypertonic mannitol had no effect on GLUT1 mRNA levels.TGF-β1 treatment for 10 hours stimulated 2DOG uptake, both in 5.5 mmol/L and 20 mmol/L glucose medium, by approximately 4.28-fold in a dose-dependent manner (2 ng/ml maximum).Kinetic analysis of 2DOG uptake revealed an increase in V(max) and a decrease in K(m) in the presence of TGF-β1. TGF-β1 also up-regulated the expression of GLUT1 mRNA in mesangial cells.The addition of anti-TGF-β neutralizing antibody (30 μg/ml) in mesangial cells cultured in enriched glucose medium (20 mmol/L) led to a 40% decrease in 2DOG uptake.Conclusions The expression of GLUT1 can be suppressed by exposure of mesangial cells to high glucose medium, which may serve as a protective mechanism against possible adverse effects of excessive glucose flux into cells.TGF-β1 stimulates glucose uptake by enhancing the expression and function of GLUT1 in mesangial cells.This effect is independent of the glucose milieu in the cultured medium.     

Effects of glucose and insulin on the H9c2 （2-1） cell proliferation may be mediated through regulating glucose transporter 4 expression

Background The change of glucose transporter 4 （GLUT4） expression could influence glucose uptake in the myocardial cells and then effect myocardial metabolism, which maybe one of the factor for the diabetes cardiovascular disease. This study aimed to explore the influence of glucose and insulin at different concentrations on H9c2 （2-1） cell proliferation and its GLUT4 expression in vitro, and evaluate the correlation between myocardial cells proliferation and GLUT4 expression. This might be helpful for understanding the relationship between glucose metabolism and cardiovascular disease. Methods According to glucose concentrations in culture medium, cultured H9c2 rat myocardial cells were divided into five groups： control group （NC, glucose concentration 5.0 mmol/L）, low glucose group （LG, glucose concentration 0.1 mmol/L）, high glucose group 1 （HG1, glucose concentration 10 mmol/L）, high glucose group 2 （HG2, glucose concentration 15 mmol/L）, high glucose group 3 （HG3, glucose concentration 20 mmol/L）. Then according to different insulin concentrations in culture medium, each group was further divided into two subgroups： normal insulin subgroup （INSc, insulin concentration 3.8 mU/L）, high insulin subgroup （INSh, insulin concentration 7.6 mU/L）. H9c2 （2-1） cells were cultured for 1, 2, 3 days, the proliferation of cells were assayed by cell counting Kit-8 assay, the expressions of GLUT4 mRNA and protein were detected with RT-PCR and Western Blotting technique, and the relation between myocardial cells proliferation and GLUT4 expression was evaluated.     

Genistein reverses free fatty acid-induced insulin resistance in HepG2 hepatocytes through targeting JNK

This study investigated the effects and molecular mechanisms of genistein in improving insulin resistance induced by free fatty acids (FFAs) in HepG2 hepatocytes. A model of insulin resistance in HepG2 cells was established by adding palmitic acid (0.5 mmol/L) to the culture medium and the cells were treated by genistein. Glucose consumption of HepG2 cells was determined by glucose oxidase method. The levels of c-jun N-terminal kinase (JNK) phosphorylation, insulin receptor substrate-1 (IRS-1) Ser307 phosphorylation, JNK, IRS-1, phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 1 (GLUT1) proteins were detected by Western blotting. The results showed that after the treatment with palmitic acid for 24 h, the insulin-stimulated glucose transport in HepG2 cells was inhibited, and the glucose consumption was substantially reduced. Meanwhile, the expressions of IRS-1, PI-3K p85 protein and GLUT1 were obviously reduced, while the levels of JNK phosphorylation and IRS-1 Ser307 phosphorylation and the expression of JNK protein were significantly increased, as compared with cells of normal control. However, the aforementioned indices, which indicated the existence of insulin resistance, were reversed by genistein at 1-4 μmol/L in a dose-dependent manner. It was concluded that insulin resistance induced by FFAs in HepG2 hepatocytes could be improved by genistein. Genistein might reverse FFAs-induced insulin resistance in HepG2 cells by targeting JNK.     

Shp-2／NF-κB Pathway Mediates the Inhibition of Lipoxin A4 onIL-1β-induced Synthesis of IL-6 in Glomerular Mesangial Cells

Objective: To examine whether lipoxin A4 (LXA4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β with or without preincubation with LXA4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzymelinked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2( SH2 ) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results:IL-1β- snulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA4 in a dose-dependent manner. LXA4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β Conclusion: LXA4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cellsthrough the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.     

EFFECTS OF RAPAMYCIN ON INTRACELLULAR CHOLESTEROL HOMEOSTASIS OF GLOMERULAR MESANGIAL CELL IN THE PRESENCE OF INTERLEUKIN-1β

Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms. Methods Intracellular cholesterol accumulation was measured by Oil Red O staining and high performance liquid chromatography. The effects of rapamycin on interleukin-1β (IL-1β)-induced mRNA and protein changes of low-density lipoprotein receptor (LDLR) and ATP-binding cassette transporter A1 (ABCA1) were assayed by quantitative real-time PCR and Western blot. Transient expressions of 3 types of mammalian target of rapamycin (mTOR), including mTOR-WT (wild type), mTOR-RR (rapamycin resistant, with kinase activity), and mTOR-RR-KD (rapamycin resistant, without kinase activity), were obtained by plasmid transfection. Results Rapamycin had no significant influence on intracellular cholesterol concentration under normal condition, but it significantly decreased the intracellular cholesterol concentration in the presence of IL-1β. Rapamycin dose-dependently suppressed the increased expression of LDLR induced by IL-1β and up-regulated the suppressed expression of ABCA1 caused by IL-1β. Transient expression of 3 types of mTOR all reduced ABCA1 mRNA expression significantly, which all could be overroded by rapamycin. Conclusions Rapamycin may contribute to the maintaining of glomerular mesangial cell intracellular cholesterol homeostasis under inflammatory state by both reducing cholesterol uptake and increasing cholesterol efflux. And the effect may be not completely mediated by mTOR.     

EXPRESSION OF PEDF mRNA AND PROTEIN IN NORMAL MOUSE RETINA AND EXPERIMENTAL CHOROIDAL NEOVASCULARIZATION TISSUES

Objective To study the expression of pigment epithelium derived factor (PEDF) in normal mouse retina and experimental choroidal neovascularization (CNV) tissues. Methods CNV mouse models were induced by diode laser. The expression of PEDF mRNA and protein in normal mouse retina and CNV tissues were detected by in situ hybridization and immunohistochemical study. Results In normal mouse retina, PEDF mRNA was observed in the ganglion cell layer, inner nuclear layer and RPE cell layer, and PEDF protein was observed mainly in the nerve fiber layer, ganglion cell layer, photoreceptor cell layer and RPE cell layer, and lower level expression of PEDF protein was also observed in the inner plexiform layer and outer plexiform layer. In CNV tissues, the expression of PEDF mRNA and protein was also observed. 3d and 1 week after photocoagulation, the expression level of PEDF was relatively lower, and increased following the development of CNV. The level was the highest 2 weeks after photocoagulation, then decreased at 3 weeks. Conclusion PEDF was expressed in different layers of retina and was obviously expressed in the CNV tissues induced by laser photocoagulation. These findings suggest that PEDF may participate and modulate the development of CNV.     

Dynamic expression of glucose transporters 1 and 3 in the brain of diabetic rats with cerebral ischemia reperfusion

Background Blood glucose control improves the outcome of diabetic patients with stroke, but the target range of blood glucose control remains controversial. The functional recruitment of ischemia penumbra is extremely important to the recovery after stroke. The present study aimed to explore the expression of brain-type glucose transporters （GLUT1 and GLUT3） in cerebral ischemic penumbra at different blood glucose levels and different ischemic-reperfusion time in diabetic hypoxia-ischemia rats. The results might provide an experimental basis for clinical treatment of diabetic patients with stroke. Methods The Wistar rats included in this study were randomly assigned to 4 groups （50 rats each）： normal control group （NC）, uncontrolled diabetic group （DM1）, poorly-controlled diabetic group （DM2）, and well-controlled diabetic group （DM3）. Diabetic rats were induced by single intraperitoneal injection of streptozotocin, and the focal ischemic rat model of middle artery occlusion （MCAO） was made by insertion of fishing thread in 6 weeks after the establishment of the diabetic model. Each group was divided into 5 subgroups （10 rats each）： four focal ischemic subgroups at different ischemic-reperfusion time （at 3,12, 24 and 72 hours after reperfusion, respectively） and one sham-operated subgroup. The mRNA and protein expression of GLUT1 and GLUT3 was assessed by RT-PCR and Western blotting, respectively. Results There was significant difference in the mRNA expression of GLUT1 and GLUT3 between the four focal ischemic subgroups and the sham-operated subgroup at different reperfusion time in each group. The mRNA expression of GLUT1 and GLUT3 in the 4 ischemic groups began to increase at 3 hours, peaked at 24 hours after reperfusion and maintained at a higher level even at 72 hours compared with that of the sham-operated subgroup. The mRNA expression of GLUT1 increased more significantly than that of GLUT3. The mRNA expression of GLUT1 and GLUT3 was significantly different between the diabetic groups and normal control group. The mRNA expression of GLUT1 and GLUT3 was increased more significantty in the diabetic groups than that in the normal control group. There was a significant difference in the mRNA expression in the groups with different blood glucose levels. The mRNA expression tended to decrease with increased blood glucose levels. The expression trend of GLUT1 and GLUT3 protein was similar to that of GLUT1 and GLUT3 mRNA. Conclusions GLUT1 and GLUT3 expression was notably up-regulated in the penumbra region after cerebral ischemia in this study. But the up-regulated amplitude of GLUT1 and GLUT3 in the diabetic rats with cerebral ischemic injury became smaller than that of the normal controls. In the treatment of diabetic patients with cerebral embolism, blood glucose control should not be too strict, otherwise the up-regulation of GLUT1 and GLUT3 induced by cerebral ischemic injury might not be able to meet the needs of energy metabolism in cells. Chin Med J 2009; 122（ 17）： 1996-2001     

EFFECTS OF RAPAMYCIN ON INTRACELLULAR CHOLESTEROL HOMEOSTASIS OF GLOMERULAR MESANGIAL CELL IN THE PRESENCE OF INTERLEUKIN-1β

Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms.
Methods Intracellular cholesterol accumulation was measured by Oil Red O staining and high performance liquid chromatography. The effects of rapamycin on interleukin-1β（1L-1β）-induced mRNA and protein changes of low-density lipoprotein receptor （LDLR） and ATP-binding cassette transporter Al （ABCAl） were assayed by quantitative real-time PCR and Western blot. Transient expressions of 3 types of mammalian target of rapamycin （mTOR）, including mTOR-WT （wild type）, mTOR-RR （rapamycin resistant, with kinase activity）, and mTOR-RR-KD （rapamycin resistant, without kinase activity）, were obtained by plasmid transfection.
Results Rapamycin had no significant influence on intracellular cholesterol concentration trader normal condition, but it significantly decreased the intracellular cholesterol concentration in the presence of IL-1β. Rapamycin dose-dependently suppressed the increased expression of LDLR induced by IL-1β and up-regulated the suppressed expression of ABCAl caused by IL-1β Transient expression of 3 types of mTOR all reduced ABCAl mRNA expression significantly, which all could be overroded by rapamycin.
Conclusions Rapamycin may contribute to the maintaining of glomerular mesangial cell intracellular cholesterol homeostasis under inflammatory state by both reducing cholesterol uptake and increasing cholesterol effiux. And the effect may be not completely mediiated by mTOR.     

Expression of Serum and Glucocorticoid-inducible Kinase1 in Diabetic Rats and Its Modulation by Fluvastatin

The expression of serum and glucocorticoid-induced protein kinase in the renal cortex of diabetic rats was examined, and the function of signal transduction mediated by SGK1 in diabetic nephropathy and its modulation by fluvastatin were also investigated. 24 male Wistar rats were randomly divided into normal control group （n = 8）, diabetic nephropathy group （n = 8） and fluvastatin-treated diabetic nephropathy group （15 mg/kg/d, n=8）. The metabolic parameters were measured at the 8th week. The expression of transforming growth factor β1 （TGF-β1） and fibronectin （FN） was immunohistochemically examined. The expression of SGK1 was detected by RT-PCR and Western blot, and CTGF mRNA was assessed by RT-PCR. As compared to DN, blood glucose, 24-h urinary protein, Cer and kidney weight index were all decreased and the weight was increased obviously in group F. At the same time, mesangial cells and extracellular matrix proliferation were relieved significantly. The levels of cortex SGK1 mRNA and protein were up-regulated, and both TGF-β1 and FN were down-regulated by fluvastatin. The mRNA of SGK1 was positively correlated with the CTGF, TGF-β1 and FN. SGK1 expression is markedly up-regulated in the renal cortex of DN group and plays an important role in the development and progress of diabetic nephropathy by means of signal transduction. Fluvastatin suppressed the increased SGKlmRNA expression in renal cortex and postponed the development of diabetic nephropathy.     

IDENTIFICATION OF GLUCOSE TRANSPORTER-1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

INTRODUCTION Glomerular mesangial cells play a key role in the development of diabetic glomerular lesions. There is a close correlation between expansion of the mesangium of the glomerulus and the clinical manifestations of diabetic nephropathy(1). Although it has become evident that the accumulation of extracellular matrix (ECM) occurs in the diabetic glomerulus and that mesangial cells in culture demonstrate enhanced production of ECM in response to elevated glucose levels (2), the me…     

Effect of rhein on glucose transporter-1 expression and its function in glomerular mesangial cells

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转化生长因子大黄酸对肾小球系膜细胞葡萄糖转运蛋白功能的影响

目的 对人肾小球系膜细胞葡萄糖转运蛋白－１（ＧＬＵＴ１）进行鉴定。研究ＧＬＵＴ１的作用特点,ＴＧＦ－β１对其影响以及大黄酸的干预作用。方法 分别用ＲＴ－ＰＣＲ,免疫荧光染色,流式细胞仪和［＾３Ｈ］－２脱氧葡萄糖摄入率,对系膜细胞ＧＬＵＴ１ｍＲＮＡ表达,蛋白质分布和功能进行鉴定。观察不同浓度ＴＧＦ－β１在加或不加大黄酸的情况下对系膜细胞葡萄糖摄入以及ＧＬＵＴ１ｍＲＮＡ表达的影响。结果 人类肾小球系膜     

犬低血流心肌缺血诱导GLUT1基因表达增加

目的：通过检测低血流心肌缺血后心肌细胞葡萄糖转运子1(GLUT1)基因的表达,探讨心肌细胞对葡 萄糖摄取增加的代谢机制。方法：建立犬低血流心肌缺血模型,放射 性核素标记方法检测心肌葡萄糖摄取量和GLUT1数量,采用Northern印迹法分析缺血心肌GLU T1 mRNA表达,采用免疫印迹法分析心肌GLUT1多肽表达。结果：与正 常心脏比较,低血流心肌缺血后,缺血心肌GLUT1 mRNA和GLUT1多肽表达明显增加,分别为 正常心肌的3.6倍和1.6倍(P<0.01)。无论在正常或缺血心脏,GLUT1表达均无部位差异 。结论：心肌缺血能诱导缺血心肌局部GLUT1表达增加,致使心肌细胞 在低血流心肌缺血过程中葡萄糖摄取和代谢增强。GLUT1表达增强可能是一种重要的心肌缺 血后代偿性保护机制。     

CV-1细胞中固醇调节元件结合蛋白-1c对Ⅰ型葡萄糖载体mRNA稳定性及蛋白表达的影响

[目的]探讨在CV-1细胞中固醇调节元件结合蛋白-1c(SREBP-1c)对Ⅰ型葡萄糖载体mRNA稳定性和蛋白表达的影响.[方法]利用DNA克隆技术,将SREBP-1c和其阴性表达体(ADDN)DNA序列重组到表达载体pSV-sport上.用SREBP-1c转染CV-1细胞,应用Northern Blot方法观察SREBP-1c对Ⅰ型葡萄糖载体mRNA的表达及其稳定性的影响,应用Western Blot方法观察SREBP-1c对Ⅰ型葡萄糖载体蛋白表达的影响.[结果]SREBP-1c可增加Ⅰ型葡萄糖载体的mRNA表达水平及稳定性,可提高Ⅰ型葡萄糖载体的蛋白表达水平.[结论]SREBP-1c可增强Ⅰ型葡萄糖载体的蛋白表达,可能通过调节Ⅰ型葡萄糖载体的表达水平而参与胰岛素对血糖的调节过程.     

2型糖尿病肾病患者肾小球系膜细胞表型及功能改变

目的研究2型糖尿病肾病系膜细胞表型和功能的改变,进一步探讨糖尿病肾病的发病机理.方法经肾活检从2型糖尿病肾病患者获取肾组织,体外培养系膜细胞.采用流式细胞术、3H-胸腺嘧啶掺入及细胞倍增时间观察细胞表型改变.系膜细胞α-平滑肌肌动蛋白(α-SMA)、层粘连蛋白、纤维连接蛋白的表达用免疫荧光染色及流式细胞仪检测.用2-脱氧-3H-葡萄糖(2-DG)测定细胞葡萄糖摄入.Northern杂交和流式细胞仪检测葡萄糖转运蛋白1(GLUT1)的表达.细胞谷氨酰胺6-磷酸果糖转氨酶(GFAT)的活性采用比色法测定.结果 2型糖尿病肾病来源的系膜细胞较正常对照表现出细胞体积增大、RNA/DNA比值增加并伴细胞增殖加快,细胞骨架蛋白α-SMA和细胞外基质合成增加.糖尿病肾病系膜细胞的葡萄糖摄入率高于对照(1 592 cpm·105 cell-1 与 1 275 cpm·105 cell-1,P<0.05),同时伴GLUT1mRNA及蛋白质表达增加.此外,糖尿病肾病系膜细胞的GFAT活性明显增高.结论 2型糖尿病肾病系膜细胞具有明显的表型与功能改变.此外,糖尿病肾病患者系膜细胞还表现出细胞糖摄入及己糖胺通路活性增加.上述表型及功能改变可能是糖尿病肾病系膜细胞病变形成的基础.     

葡萄糖对丁酸钠诱导的结肠癌细胞增殖抑制和凋亡效应的调节作用及其可能机制

目的:探讨葡萄糖对丁酸钠诱导的结肠癌细胞增殖抑制和凋亡效应的影响,并探讨其可能机制。方法:用MTT法检测细胞存活率。细胞凋亡用TUNEL法检测。用RT-PCR法检测葡萄糖转运蛋白1(GLUT1)、单羧酸载体1(MCT1)的mRNA表达水平。结果:低浓度葡萄糖诱导HT-29细胞系凋亡、调节其增殖,且葡萄糖能够明显抵制丁酸钠诱导凋亡和增殖抑制作用,同时对GLUT1 mRNA、MCT1 mRNA也有一定抑制作用。结论:葡萄糖浓度变化能够明显影响丁酸钠的诱导凋亡和增殖抑制作用,这种影响可能与细胞内葡萄糖和丁酸钠的浓度有关。     

高糖刺激对体外培养大鼠肾小球系膜细胞表达TGF-β_1和PDGF-BB的影响

目的探讨高糖刺激对体外培养的大鼠肾小球系膜细胞(HBZY-1)表达转化生子因子(TGF-β1)和血小板源性生长因子(PDGF-BB)的影响。方法体外培养大鼠肾小球系膜细胞,传代后分为正常组(葡萄糖浓度为5.6mmol/L)、高糖A组(葡萄糖浓度为15mmol/L)、高糖B组(葡萄糖浓度为30mmol/L),分别培养12h、24h、48h后,应用逆转录聚合酶链反应(RT-PCR)测定各组细胞TGF-β1和PDGF-BB mRNA的表达水平,应用免疫细胞化学法检测各组细胞中TGF-β1和PDGF-BB蛋白的表达。结果①正常组细胞可表达TGF-β1及PDGF-BB;②与正常组相比,高糖各组细胞TGF-β1和PDGF-BB的mRNA及蛋白表达均升高(P<0.01);③与高糖A组比较,高糖B组系膜细胞中TGF-β1和PDGF-BB的mRNA及蛋白表达均升高(P<0.01)。结论高糖刺激可以上调大鼠肾小球系膜细胞TGF-β1和PDGF-BB的表达。