NYGGF4基因过表达对脂肪细胞胰岛素敏感性及分泌功能的影响

目的：观察肥胖相关新基因NYGGF4过表达对脂肪细胞的胰岛素敏感性及分泌功能的影响。方法：体外培养稳定转染NYGGF4基因（NYGGF4-pcDNA3.1）的3T3-L1前体脂肪细胞,以转染空载体（pcDNA 3.1）的3T3-L1细胞为对照,胰岛素加地塞米松加1-甲基-3-异丁基黄嘌呤(MDI)方案诱导细胞分化成熟,液闪仪测定3H标记的葡萄糖摄取率,Western blot检测葡萄糖转运体4（GLUT4）的表达及转位;采用ELISA双抗体夹心法检测培养上清中TNF-α、IL-6、脂联素、抵抗素4种细胞因子的水平。结果：NYGGF4过表达显著降低胰岛素刺激的葡萄糖摄取率（P<0.05）。NYGGF4过表达对总的GLUT4的表达量没有影响,但明显降低胰岛素刺激的GLUT4由细胞浆向细胞膜的转位（P<0.05）。过表达NYGGF4的脂肪细胞其培养上清中TNF-α、IL-6、脂联素及抵抗素的分泌水平与对照组相比差异无显著性（P>0.05）。结论：NYGGF4基因过表达通过下调胰岛素刺激的GLUT4的转位而减少脂肪细胞的葡萄糖摄取率,提示脂肪细胞对胰岛素的敏感性降低,而对脂肪细胞的分泌功能无明显影响。［中国当代儿科杂志,2009,11（10）：846-849］     

重组人白细胞介素-6对脂肪细胞蛋白质酪氨酸磷酸酶1B、白细胞介素-6及抵抗素基因表达的影响

目的观察重组人白细胞介素-6(rhIL-6)对SW872脂肪细胞蛋白质酪氨酸磷酸酶1B(PTP1B)、抵抗素及IL-6基因表达的影响,探讨rhIL-6对脂肪细胞分泌功能的调节作用。方法体外培养,油酸诱导SW872前脂肪细胞分化为成熟的脂肪细胞,在培养液中加入不同水平rhIL-6(0、1、5、10、20、50μg/L)作用24 h,加入20μg/L rhIL-6后分别作用不同时间(0、4、8、12、24 h)。收集细胞提取总RNA,采用半定量反转录酶聚合酶链反应方法检测SW872脂肪细胞PTP1B、抵抗素、IL-6 mRNA水平。结果rhIL-6 1μg/L作用24 h,对SW872脂肪细胞IL-6 mRNA的表达无影响;随着rhIL-6水平的增加,SW872脂肪细胞IL-6 mRNA的表达水平逐渐增加,但以20μg/L rhIL-6的作用最强(F=233.9 P<0.01);20μg/L rhIL-6作用4 h即可促进SW872脂肪细胞IL-6 mRNA的表达,随着作用时间的延长,其促进作用更加明显(F=247.8 P<0.01)。1μg/L rhIL-6作用24 h,对SW872脂肪细胞PTP1B mRNA的表达无影响;5μg/L rhIL-6即可促进SW872脂肪细胞PTP1B mRNA表达,50μg/L rhIL-6作用24 h,对PTP1B mRNA的表达促进作用更明显(F=515.58 P<0.01);20μg/L rhIL-6作用4 h对脂肪细胞PTP1B mRNA的表达无影响,作用8 h即可促进PTP1BmRNA的表达,随着作用时间的延长其作用更加明显(F=498.62 P<0.01)。不同水平、不同作用时间下,rhIL-6对SW872脂肪细胞抵抗素mRNA的表达无明显影响(F=9.6,10.5 Pa>0.05)。结论rhIL-6以剂量和时间相关的方式促进SW872脂肪细胞PTP1B及IL-6 mRNA表达,对抵抗素mRNA的表达无影响。     

高脂饮食大鼠血清抵抗素水平及下丘脑细胞因子信号转导抑制因子-3 基因表达的变化

目的观察高脂饮食大鼠血清抵抗素水平及下丘脑细胞因子信号转导抑制因子-3(SOCS-3)基因表达变化及其关系,探讨高脂饮食引发肥胖的发生机制。方法雄性6周龄SD大鼠16只。体质量80～100 g。随机分为基础饮食组和高脂饮食组,每组8只。单笼饲养,自由饮食饮水,明暗周期12 h,环境温度21～23℃,每周称体质量、测体长和尾长1次。大鼠饲养9周后,以乌拉坦(0.4 g/kg)深度麻醉,取心脏全血离心,采用ELISA法检测其血清抵抗素水平,葡萄糖氧化酶法检测其血清葡萄糖水平。取其下丘脑组织100～120 mg,用Trizol一步法提取总mRNA,经电泳显示28、18、5 S清晰条带,测定A260/A280>1.8。应用反转录聚合酶链反应(RT-PCR)检测其SOCS-3 mRNA表达水平。PCR产物经18 g/L琼脂糖凝胶电泳后,利用凝胶图像分析系统测定电泳条带密度值,以甘油醛-3-磷酸脱氢酶(GAPDH)为内参照,计算SOCS-3 mRNA相对水平,结果以SOCS-3条带占GAPDH条带密度百分率(%)表示。结果1.高脂饮食组大鼠体质量、体长、尾长及Lee′s指数明显高于基础饮食组(Pa<0.001);2.高脂饮食组大鼠血清血糖及抵抗素水平明显升高,与基础饮食组比较均有显著性差异(Pa<0.01);3.高脂饮食组大鼠下丘脑SOCS-3 mRNA表达水平显著高于基础饮食组(P<0.05);4.二组大鼠血清抵抗素水平与下丘脑SOCS-3 mRNA表达水平无明显相关性(Pa>0.05)。结论高脂饮食可导致大鼠血清抵抗素水平升高,进而诱导SOCS-3基因表达,在肥胖、抵抗素所致的胰岛素抵抗过程中SOCS-3可能发挥了重要的介质作用。     

肥胖儿童血清抵抗素与胰岛素抵抗、β细胞功能的关系

目的研究不同程度肥胖儿童血清抵抗素与胰岛素抵抗(IR)、胰岛β细胞功能的关系。方法根据体质量指数(BMI)将研究对象分为正常对照组(BMI<22,n=30)、肥胖1组(23≤BMI<30,n=82)和肥胖2组(BMI≥30,n=31)。对3组患儿进行葡萄糖耐量试验,测量空腹血清抵抗素,分别采用稳态模型的胰岛素抵抗指数(HOMAIR)、胰岛β细胞功能指数(HOMAβ)和总体胰岛素敏感指数(WBISI)、30min胰岛素增量与葡萄糖增量比值(△I30/△G30)作为基础和糖负荷后胰岛素敏感性和胰岛β细胞功能的评价指标。结果肥胖儿童糖耐量减低6例(5.3%),2型糖尿病2例(1.8%)。随BMI增加,糖负荷后2h血糖(2hPG)、血糖曲线下面积(AUCg)、空腹胰岛素(FINS)、胰岛素曲线下面积(AUCi)和HOMAIR逐步增高(P均<0.05),WBISI逐步降低(P<0.001);肥胖组HOMAβ和△I30/△G30较对照组明显上升(P均<0.05);肥胖组间无显著差异(P均>0.05);空腹血清抵抗素3组间差异无显著性意义(P>0.05);8例糖耐量异常儿童抵抗素较对照组略高,但无显著差异(P>0.05)。BMI与2hPG、FINS、HOMAIR、HOMAβ、△I30/△G30呈明显正相关(P均<0.05),与WBISI呈高度负相关(P<0.001),抵抗素与上述指标及BMI无明显相关(P均>0.05)。结论肥胖儿童存在IR、糖负荷后血糖水平升高和胰岛β细胞分泌功能上调,随肥胖程度加重,胰岛素敏感性进一步降低,而胰岛β细胞分泌功能无相应增强。循环抵抗素水平可能不是介导儿童肥胖及IR的关键因素,抵抗素的确切作用尚待进一步研究。     

川崎病患儿急性期血浆IFN-γ、IL-4水平变化研究

目的探讨川崎病(KD)急性期T细胞的功能状态及临床意义。方法ELISA法检测急性期KD患儿35例和10例健康儿童血浆Th1/Th2细胞的代表性细胞因子IFN-γ、IL-4水平变化,评价T细胞功能状态。结果急性期KD患儿血浆[IFN-γ(34±17)ng/L、IL-4(45±23)ng/L]水平较对照组[IFN-γ(59±21)ng/L、IL-4(69±28)ng/L]均明显下降,差异有显著性(P<0.05);两组IFN-γ/IL-4值比较,差异无显著性(P<0.05)。冠状动脉受累(CA)组[IFN-γ(28±15)ng/L、IL-4(31±14)ng/L]与无冠状动脉受累(NCA)组[IFN-γ(39±25)ng/L、IL-4(43±21)ng/L]比较,差异无显著性(P>0.05);同样,两组IFN-γ/IL-4值比较,差异无显著性(P>0.05)。结论KD急性期T细胞功能受抑,这或许是KD免疫学特征之一。     

Resistin结合多肽对胰岛β细胞株RINm5F分泌功能的影响

目的 先前研究已显示resistin结合多肽(RBP)能拮抗resistin对脂肪细胞脂质代谢和内分泌功能的调节作用.本研究试图阐明RBP对胰岛β细胞株RINm5F分泌功能的影响.方法 以不同水平RBP(10-10,10-11,1012mol/L)与RINm5F共培养30min、60min、2h后取上清,ELISA法测其胰岛素,RT-PCR法检测细胞中葡萄糖转运子2(GLUT2)mRNA表达水平,Western blotting法检测细胞中GLUT2的蛋白水平,并采用FURA-3/AM钙荧光染色法检测胰岛素分泌的重要启动因素:细胞内钙水平.结果 RBP在10-12mol/L时干预2h,不影响胰岛β细胞株RINm5F的细胞活性.但能显著刺激胰岛素分泌,RBP为10-12mol/L时干预2h,细胞内钙明显增多.在Resistin刺激下,GLUT2 mRNA水平和蛋白水平均显著上调.结论 RBP能促进RINm5F细胞株胰岛素的分泌,其可能机制是RBP上调了GLUT2的表达,进而增加了细胞内钙的水平,最终导致胰岛素分泌增加.     

维生素D对高糖诱导足细胞胰岛素抵抗的保护作用

目的观察维生素D(VitD)对高糖诱导足细胞胰岛素抵抗的保护作用及其机制。方法体外分化培养的小鼠永生化足细胞随机分为:足细胞+5 mmol/L葡萄糖(A组),足细胞+5 mmol/L葡萄糖+1 nmol/L丙二醇(B组),足细胞+30mmol/L葡萄糖+1 nmol/L丙二醇(C组),足细胞+30 mmol/L葡萄糖+1 nmol/L丙二醇+1 nmol/L VitD(D组)。培养48 h测定足细胞凋亡百分率,MTT法测定足细胞活性,RT-PCR测定足细胞维生素D受体(VDR)及胰岛素受体底物蛋白1(IRS1)的表达,Western blotting检测IRS1、肌醇依赖蛋白激酶(Akt)、细胞外调节蛋白激酶(ERK1/2)的表达及其磷酸化水平。结果 4组间足细胞凋亡及活力,VDR和IRS1基因表达水平,信号蛋白IRS1、ERK1/2磷酸化水平的差异均有统计学意义(P均0.05),Akt磷酸化水平的组间差异无统计学意义(P0.05)。与A、B和D组相比,C组足细胞凋亡百分率升高,足细胞活力降低,VDR和IRS1 mRNA表达下调,IRS1磷酸化水平下降,ERK1/2磷酸化水平升高,差异均有统计学意义(P均0.05);而A、B、D三组间p-IRS1/IRS1、p-ERK1/2/ERK1/2水平差异均无统计学意义(P0.05)。结论 VitD-VDR系统减轻高糖诱导的足细胞凋亡,活化胰岛素信号通路,拮抗胰岛素抵抗信号,改善足细胞胰岛素抵抗。     

2E8-Genistein免疫毒素靶向杀伤Nalm-6白血病细胞作用及其机制研究

目的 观察2E8(自制鼠抗人CD19新单克降抗体)-4',5',7-三羟基异黄酮(Genistein,Gen)免疫毒素(2E8-Gen)体外靶向杀伤Nalm-6细胞作用及其机制,为靶向治疗B细胞系自血病、淋巴瘤奠定基础.方法 通过细胞形态观察、台盼蓝拒染法、细胞生长曲线比较、MTT比色法和流式细胞术.结果作用24 h,2E8-Gen对Nalm-6细胞具有明显的杀伤作用,浓度在20～100 nmol/L 9个观测点的细胞存活率[从20 nmol/L时的(71.8±7.9)%逐步降到100 nmol/L时的(16.6±12.9)%,n=3]与对照组[(100±13.9)%]相比差异均有统计学意义(P<0.05),浓度在80 nmol/L以上杀伤作用显著加强;100 nmol/L 2E8-Gen在24、48、72 h对Nalm-6细胞生长抑制率分别为82%、84%、94%,呈时间依赖关系.100 nmo/L浓度2E8-Gen组的Nalm-6细胞生长曲线与空白组、PBS组和相同浓度的纯2E8组的Nalm-6细胞生长曲线相比,差异有统计学意义(F=152.15,P=2.15×10-7),但后3组间差异无统计学意义(F=1.51,P=0.29);而100 nmol/L 2E8-Gen对CD19阴性的Molt-3细胞的生长曲线与空白组相比无明显影响(F=0.34,P=0.59).24 h 100 nmol/L 2E8-Gen组早期凋亡Nalm-6细胞阳性率[(33.45±8.77)%]明显高于对照组[(10.44±1.28)%,t=-4.39,P=0.001].结论 2E8-Gen对有CD19抗原表达的Nalm-6细胞具有良好的选择性杀伤作用,并呈时间依赖关系,浓度在80 nmol/L以上杀伤作用显著加强,凋亡机制参与了该杀伤作用.     

慢性活动性EB病毒感染外周血淋巴细胞免疫亚群变化特征的研究

目的 研究慢性活动性EB病毒感染(CAEBV)宿主细胞免疫功能的变化.方法 应用流式细胞仪检测2004年3月至2008年4月住院的CAEBV患儿、急性EBV感染(acute EBVinfection,AEBV)患儿以及正常儿童外周血淋巴细胞免疫亚群.结果 CAEBV组外周血白细胞[3325×106/L,中位数(下同)]、淋巴细胞(1078×106/L)、NK细胞(68×106/L)、B细胞(84×106/L)、总T细胞(684×106/L)、CD4+T细胞(406×106/L)和CD8+T细胞(295×106/L)计数均高于AEBV组(P<0.05).CAEBV组CD4+功能、亚群的比例(94.5%)低于正常组(98.7%)(P<0.05),但高于AEBV组(74.0%)(P<0.05);而CD8+功能哑群的比例(40.7%)与正常组(48.3%)比较差异无统计学意义,但高于AEBV组(21.0%)(P<0.05).CAEBV组的调节亚群比例(5.0%)虽高于正常组(4.6%)(P<0.05),但低于AEBV组(5.8%)(P<0.05).CAEBV组初始T细胞比例(32.3%/37.5%)低于正常组(58.3%/56.6%)(P<0.05),其效应记忆T细胞的比例(23.9%/15.1%)低于较AEBV组(36.5%/69.8%)(P<0.05),而CD8+假初始T细胞(17.5%)的比例高于其他两组(12.0%和9.2%)(P<0.05).CAEBV组CD8+激活亚群(84.4%/34.0%)高于正常组(44.1%/16.7%)(P<0.05),但低于AEBV组(96.0%/95.0%)(P<0.05).结论 CAEBV患儿体内存在淋巴细胞亚群失衡和细胞免疫功能紊乱,可能与CAEBV的慢性活动性有关.检测外周血淋巴细胞亚群有助于CAEBV的诊断和鉴别诊断.     

阿奇霉素对支气管哮喘患儿外周血辅助性T淋巴细胞功能的影响

目的 探讨阿奇霉素对支气管哮喘患儿外周血辅助性T淋巴细胞(Th)1/Th2功能的影响.方法 选取24例支气管哮喘发作期患儿为支气管哮喘组,20例健康儿童为健康对照组.二组均在无菌条件下获取外周血单个核细胞(PBMC)悬液,哮喘组予不同质量浓度(0.2、0.1、0.05、0mg/L)阿奇霉素治疗,健康对照组未予阿奇霉素干预,均体外培养48h.酶联免疫吸附(ELISA)法检测培养上清液中干扰素-γ(IFN-γ)、IL-4及IL-10水平变化.采用SPSS ll.5软件进行统计学分析.结果 l.支气管哮喘组患儿PBMC产生IL-4水平明显高于健康对照组(P<0.05),IFN-γ/IL-4比值明显低于健康对照组(P<0.05),IFN-γ和IL-10水平与健康对照组比较无显著性差异(Pa>0.05);2.支气管哮喘组患儿PBMC在阿奇霉素0.1mg/L水平条件下,IL-4分泌水平明显高于0.2mg/L和0.05mg/L组(Pa<0.05)及空白对照组(P<0.01),各组间PBMC分泌IFN-γ水平和IFN-γIL-4比值均无显著性差异(Pa>0.05);3.阿奇霉素0.2mg/L和0.1 mg/L组PBMC产生IL-10水平均明显高于空白对照组(P<0.01,0.05),0.2mg/L组明显高于0.05 mg/L组(P<0.05),其他各组间均无显著性差异(Pa>0.05).结论 常规质量浓度阿奇霉素(0.1mg/L)对支气管哮喘患儿外周血Th1/Th2平衡无明显影响,但可通过上调PBMC产生IL-10水平,而间接发挥其免疫调节作用.     

肥胖儿童血清抵抗素变化及其临床意义

目的 探讨饮食控制和运动疗法对肥胖儿章血清抵抗素水平的影响及其临床意义。方法 测定36例肥胖儿童饮食控制和运动治疗前后血清抵抗素、胆固醇（CHO）、甘油三脂（TG）、低密度脂蛋白胆固醇（LDL-C）、空腹和葡萄糖耐量试验（OGTT）2小时血糖、胰岛素，计算胰岛素抵抗指数（HOMA-IR）。结果 肥胖儿童血清抵抗素水平显著高于对照组（P〈0．05），并与OGTT2小时血糖、胰岛素呈正相关（P〈0．05）；经饮食控制和运动治疗后肥胖儿童血清抵抗素、体重、BMI、血清CHO、TG、LDL-C、空腹和OGTT2小时胰岛素、HOMA-IR显著低于治疗前（P〈0．05），治疗前后空腹和OGTT2小时血糖差异无显著性（P〉0．05）。结论 肥胖儿童血清抵抗素水平升高，并与OGTT2小时血糖、胰岛素呈正相关，推测将来有可能以检测血清抵抗素水平来了解肥胖儿童有否存在糖耐量受损；饮食控制和运动疗法可使肥胖儿童血清抵抗素降低，胰岛素抵抗减轻。     

高糖对肾小管上皮细胞Toll样受体4表达的影响及安体舒通的干预作用

目的：旨在探讨高糖对肾小管上皮细胞Toll样受体4（TLR4）表达的影响及安体舒通（spironolactone,SP）对其表达的干预作用。方法：体外培养肾小管上皮细胞（NRK-52E）,分成低糖组（5 mmol/L葡萄糖的DMEM）、高糖组(25 mmol/L葡萄糖的DMEM),SP组(25 mmol/L葡萄糖的DMEM+10-7mol/L安体舒通)。采用细胞免疫化学、RT-PCR、Western blot检测各组细胞TLR4蛋白和mRNA表达情况,同时取细胞培养上清液用ELISA法检测白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）水平。结果：高糖组培养6 h TLR4 mRNA出现表达增高,24 h仍高于低糖组;SP组TLR4 mRNA明显低于同时期高糖组的表达,但仍高于低糖组。高糖组培养24 h TLR4蛋白较低糖组升高,48 h TLR4蛋白表达进一步升高;SP组TLR4蛋白表达明显低于同时期高糖组表达,但仍高于低糖组。高糖组IL-6、TNF-α较低糖组明显升高;而SP组IL-6、TNF-α高于低糖组,但明显低于高糖组。结论：高糖促使NRK-52E细胞的TLR4的表达上调,TNF-α、IL-6等炎性因子表达升高,TLR4参与了糖尿病肾病的微炎症反应;安体舒通可通过下调TLR4表达水平,减少相关炎症介质的生成,这可能是其在糖尿病肾病中具有肾脏保护作用的机制之一。［中国当代儿科杂志,2010,12（4）：280-283］     

Adolescents with clinical type 1 diabetes display reduced red blood cell glucose transporter isoform 1 (GLUT1)

Type 1 diabetic (T1D) adolescent children on insulin therapy suffer episodes of both hyper‐ and hypoglycemic episodes. Glucose transporter isoform GLUT1 expressed in blood–brain barrier (BBB) and red blood cells (RBC) compensates for perturbed circulating glucose toward protecting the supply to brain and RBCs. We hypothesized that RBC‐GLUT1 concentration, as a surrogate for BBB‐GLUT1, is altered in T1D children. To test this hypothesis, we measured RBC‐GLUT1 by enzyme‐linked immunosorbent assay (ELISA) in T1D children (n = 72; mean age 15.3 ± 0.2 yr) and control children (CON; n = 11; mean age 15.6 ± 0.9 yr) after 12 h of euglycemia and during a hyperinsulinemic–hypoglycemic clamp with a nadir blood glucose of ?3.3 mmol/L for 90 min (clamp I) or ?3 mmol/L for 45 min (clamp II). Reduced baseline RBC‐GLUT1 was observed in T1D (2.4 ± 0.17 ng/ng membrane protein); vs. CON (4.2 ± 0.61 ng/ng protein) (p < 0.0001). Additionally, baseline RBC‐GLUT1 in T1D negatively correlated with hemoglobin A1c (HbA1c) (R = ?0.23, p < 0.05) but not in CON (R = 0.06, p < 0.9). Acute decline in serum glucose to 3.3 mmol/L (90 min) or 3 mmol/L (45 min) did not change baseline RBC‐GLUT1 in T1D or CON children. We conclude that reduced RBC‐GLUT1 encountered in T1D, with no ability to compensate by increasing during acute hypoglycemia over the durations examined, may demonstrate a vulnerability of impaired RBC glucose transport (serving as a surrogate for BBB), especially in those with the worst control. We speculate that this may contribute to the perturbed cognition seen in T1D adolescents.     

Seizures, ataxia, developmental delay and the general paediatrician: glucose transporter 1 deficiency syndrome

AIM: Glucose transporter 1 deficiency syndrome (GLUT1-DS) is an important condition for the general paediatrician's differential armamentarium. We describe a case series of eight patients in order to raise awareness of this treatable neurometabolic condition. The diagnosis of GLUT1-DS is suggested by a decreased absolute cerebrospinal fluid (CSF) glucose value (<2.2 mmol/L) or lowered CSF: plasma glucose ratio (<0.4). METHODS: This is a review of eight Queensland patients with GLUT1-DS. The clinical presentation, clinical course, laboratory investigations and treatment outcomes are discussed. RESULTS: The clinical features noted in our patient cohort include combinations of ataxia, developmental delay and a severe seizure disorder that is refractory to anticonvulsant medications. Seizures are the most common clinical manifestation and may be exacerbated by phenobarbitone. The paired CSF: plasma glucose results ranged from 0.2 to 0.39 (normal <0.6) with an average of 0.33. 3-O-Methyl-D-Glucose uptake and GLUT1 Genotyping analysis have been performed on five patients thus far. Rapid and impressive seizure control was observed in 100% of our patients once the ketogenic diet was instituted, with half of the cohort being able to wean completely from anticonvulsants. CONCLUSION: Children presenting with a clinical phenotype consisting of a refractory seizure disorder, ataxia and developmental delay should prompt the consideration of Glucose transporter 1 deficiency syndrome. While the diagnostic test of lumbar puncture is an invasive manoeuvre, the diagnosis provides a viable treatment option, the ketogenic diet. GLUT1-DS displays clinical heterogeneity, but the value of early diagnosis and treatment is demonstrated by our patient cohort.     

Inhibitory effects of diazoxide or polymyxin B on glucose transport by isolated rat erythrocytes or adipocytes

The inhibitory effects of diazoxide or polymyxin B on 3-O-methylglucose uptake were studied in isolated rat erythrocytes or adipocytes to elucidate the mechanisms of the actions of these agents. One to three mmol/L diazoxide significantly inhibited 3-O-methylglucose uptake into erythrocytes by 11–33% without altering the equilibrium space, while 0.3 mmol/L diazoxide did not. The inhibitory effect was exerted in a dose-dependent manner in this concentration range. To test whether polymyxin B affects the process of insulin action or the glucose transport activity recruited by insulin, adipocytes prestimulated with insulin and exposed to 2 mmol/L potassium cyanide (KCN) were employed since the cells, on which glucose transporters recruited by insulin were located quiescently, were useful to estimate the effect of an agent on glucose transport activity per se. Polymyxin B (100 μg/mL) inhibited the insulin-stimulated uptake activity in this transport system by 22.5% while it inhibited the insulin-stimulated uptake activity in intact adipocytes which were not exposed to KCN by 32.2%. These results suggest that diazoxide inhibits the function of the erythrocyte glucose transporter, GLUT1*** (classified by Bell et al.), and indicate that the inhibition of the glucose transport activity recruited by insulin is the major effect of polymyxin B (100 μg/mL) and the inhibition of the process of insulin action is rather small.     

Actions of peroxovanadate or tungstate on glucose transport by isolated rat adipocytes

The effects of peroxovanadate or tungstate on 3-O-methylglucose uptake were characterized using isolated rat adipocytes to elucidate the mechanism(s) of their actions. The stimulatory effect of peroxovanadate was observed from 1 μmol/L and reached the maximum at about 100 μmol/L. The concentration showing the half-maximal effect was approximately 16 μmol/L. The maximal response of peroxovanadate was 1.19 times higher than that of insulin significantly (P < 0.01). On the other hand, the stimulatory effect of tungstate was seen only at the higher concentrations of 10–30 mmol/L Judging from the experiments using different tungsten compounds, tungstic acid (WO₄^2-) appeared responsible for the effect. The effects of 20 mmol/L tungstate and 20 nmol/L insulin were not additive. The stimulatory effects of 1 mmol/L peroxovanadate, 20 mmol/L tungstate or 20 nmol/L insulin were not seen in the adipocytes deprived of ATP by exposure to 2 mmol/L KCN. The adipocytes which had been stimulated with insulin and further exposed to 2 mmol/L KCN were used to test whether or not peroxovanadate works directly on the function of glucose transporters. In such cells on which GLUT4-rich transporters were rendered immobile, the effect of peroxovanadate was not observed. These results indicate that the effects of peroxovanadate or tungstate are ATP or energy dependent and may be exerted through the mechanism analogous to that of insulin action, and suggest that peroxovanadate does not directly activate the function of GLUT4.     

TNFalpha decreases gluconeogenesis in hepatocytes isolated from 10-day-old rats

Gluconeogenesis decreases during septic shock, but its mechanism is not well known. Tumor necrosis factor alpha (TNF-alpha), which is a key cytokine in septic shock, can increase GLUT1 gene expression and glucose uptake in muscles and fatty tissues. TNF-alpha does not alter the metabolism of hepatocytes in which GLUT2 is the predominant glucose transporter. However, GLUT1 is the predominant glucose transporter in hepatocytes of 10-d-old rats. Thus, we hypothesized that TNF-alpha might increase glucose uptake and glycolysis in those cells, and decrease gluconeogenesis. In the present study, hepatocytes isolated from 10-d-old rats were incubated with TNF-alpha at the concentrations of 0, 0.98, 9.8, 98, and 980 ng/mL to evaluate TNF-alpha effects on gluconeogenesis and glucose uptake. TNF-alpha increased glucose uptake (41.1 +/- 8 to 114 +/- 21.4 micromol/10(6) cells at the concentration of 980 ng/mL of TNF-alpha) in a dose-dependent manner, and decreased gluconeogenesis (98.2 +/- 8.2 to 1.1 +/- 3.2 micromol/10(6) cells at the concentration of 980 ng/mL of TNF-alpha) in a dose-dependent manner. The decrease of glucokinase mRNA and GLUT1 mRNA abundance correlated with glucose uptake (r = 0.988 and 0.997, respectively), and the decrease of phosphoenolpyruvate carboxykinase mRNA abundance correlated with the decrease of gluconeogenesis (r = 0.972). The decrease of gluconeogenesis by TNF-alpha correlated with the increase of glucose uptake (r = -0.988). We concluded that TNF-alpha reciprocally suppressed gluconeogenesis in hepatocytes isolated from 10-d-old rats.     

Severe hyperinsulinemia, decreased GLUT3 and GLUT4 expression, and increased retinol binding protein 4 in a patient with chronic graft-versus-host disease post bone marrow transplantation

Hyperinsulinemia with or without DM2 is a frequent long-term sequela of BMT, especially following cGvHD. In this report, an extensive evaluation of a patient with cGvHD is described: glucose and insulin during OGTT, markers of inflammation, adiponectin and RBP4, body composition analysis, and the kinetics of GLUT3 and GLUT4 in circulating monocytes were evaluated. Hyperinsulinemia, associated with partial lipodystrophy, elevated RBP4, low adiponectin levels, and decreased expression of GLUT3 and GLUT4 were detected. The defects disclosed in this particular patient possibly explain, at least in part, the mechanisms underlying insulin resistance in patients undergoing BMT. It is not clear whether insulin resistance was caused by the drugs, the process itself, or the residual damage to the muscles and/or adipose tissue.