地塞米松和胰岛素调节猪脂肪细胞SOCS-3、OB、 GLUT4和PPARg 基因的表达

猪是研究糖尿病最理想的模型动物, 研究胰岛素和胰岛素抵抗是研究糖尿病的重要环节。为明确SOCS-3在胰岛素抵抗中的作用, 分别用100 nmol/L的胰岛素, 300 nmol/L的地塞米松处理原代培养的猪脂肪细胞诱导胰岛素抵抗; 利用半定量RT-PCR技术分别检测SOCS-3、OB、GLUT4和PPARg 基因表达变化。结果发现, 胰岛素增加了GLUT4、SOCS-3和PPARg 基因的表达, 对OB基因表达变化没有显著性影响; 地塞米松诱导的胰岛素抵抗状态下OB和SOCS-3基因表达水平升高, 而GLUT4和PPARγ基因表达水平显著下调。研究结果表明, GLUT4基因表达量水平的升高可能是由于PPARg的高表达引起, SOCS-3基因的不同表达水平对胰岛素信号的抑制效果不同。地塞米松诱导的胰岛素抵抗不仅表现在对葡萄糖转运的抑制, 也反映在抑制了胰岛素信号; 而SOCS-3基因可能是消除胰岛素抵抗的一个有效靶基因。     

地塞米松和胰岛素调节猪脂肪细胞SOCS-3、OB、GLUT4和PPARγ基因的表达

猪是研究糖尿病最理想的模型动物,研究胰岛素和胰岛素抵抗是研究糖尿病的重要环节。为明确SOCS-3在胰岛素抵抗中的作用,分别用100nmol／L的胰岛素,300nmol／L的地基米松处理原代培养的猪脂肪细胞诱导胰岛素抵抗;利用半定量RT-PCR技术分别检测SOCS-3、OB、GLUT4和PPARγ基因表达变化。结果发现,胰岛素增加了GLUT4、SOCS-3和PPARγ基因的表达,对OB基因表达变化没有显著性影响;地塞米松诱导的胰岛素抵抗状态下OB和SOCS-3基因表达水平升高,而GLUT4和PPARγ基因表达水平显著下调。研究结果表明,GLUT4基因表达量水平的升高可能是由于PPARγ的高表达引起,SOCS-3基因的不同表达水平对胰岛素信号的抑制效果不同。地塞米松诱导的胰岛素抵抗不仅表现在对葡萄糖转运的抑制,也反映在抑制了胰岛素信号;而SOCS-3基因可能是消除胰岛素抵抗的一个有效靶基因。     

游离脂肪酸对3T3-L1脂肪细胞糖代谢的影响

目的:通过培养3T3-L1前脂肪细胞,并诱导其分化至成熟,研究游离脂肪酸对脂肪细胞糖代谢的影响。方法:培养诱导3T3-L1脂肪细胞,用油红O染色鉴定并比较其形态结构的变化。LPS、EPA、SA、PA干预成熟脂肪细胞,收集不同时间的培养基,葡萄糖氧化酶法算出各组脂肪细胞的葡萄糖消耗量。用Western blot检测不同时间各组干预后细胞AMPK、GLUT4蛋白含量。结果:油红O染色鉴定成熟脂肪细胞胞浆中的脂滴染成红色,并出现戒环样结构;诱导分化第8天,90%以上细胞均分化成熟。含LPS、EPA、SA、PA的培养基作用于成熟脂肪细胞,随着时间的延长,显著抑制脂肪细胞对葡萄糖的吸收(P<0.05),同时,脂肪细胞AMPK、GLUT4蛋白含量在减少(P<0.05)。结论:游离脂肪酸可以诱导胰岛素抵抗的分子机制可能是通过胰岛素信号通路激活蛋白激酶(AMPK),进而影响GLUT4的蛋白表达,使脂肪细胞的葡萄糖吸收率减低,影响脂肪细胞的糖代谢。     

NF-κB p65在高糖诱导3T3-L1脂肪细胞胰岛素抵抗中的作用

建立高糖诱导胰岛素抵抗的细胞模型,研究高糖对3T3-L1脂肪细胞NF-κB p65表达及转位的影响。诱导成熟的3T3-L1脂肪细胞与5.0mmol/L的葡萄糖含或不含0.6nmol/L的胰岛素(LGIns 组与LGIns-组)或者与25.0mmol/L葡萄糖含或不含0.6nmol/L的胰岛素(HGIns 组与HGIns-组)培养18h,以2-脱氧-[3H]-D-葡萄糖摄入法观察葡萄糖的转运率,用Western印迹检测总NF-κBp65及核NF-κB p65的表达,用激光扫描共聚焦(CLSM)对NF-κB p65进行定位显示。结果显示,仅HGIns 组,即3T3-L1脂肪细胞与25.0mmol/L葡萄糖含0.6nmol/L的胰岛素培养18h后,胰岛素刺激的葡萄糖转运减少55%(P<0.01),同时Western印迹和CLSM均显示NF-κB p65核转位增加(P<0.01),但对3T3-L1脂肪细胞总NF-κB p65的表达无明显影响(P>0.05)。研究结果表明,只有在胰岛素(0.6nmol/L)存在的条件下,高糖(25.0mmol/L)才可以诱导胰岛素抵抗,其分子机制可能与其刺激NF-κB p65的核转位,调节相关基因的表达有关。     

miR-196a-5p对3T3-L1前脂肪细胞增殖和分化的影响效应

目的:探究miR-196a-5p对小鼠前体脂肪细胞增殖、分化的影响及其潜在的分子机制。方法:(1)构建小鼠肥胖模型,RT-PCR检测脂肪组织中miR-196a-5p表达量;(2)鸡尾酒法诱导3T3-L1前脂肪细胞分化,RT-PCR检测分化过程中miR-196a-5p的表达变化;(3)合成miR-196a-5p mimics和inhibitors转染3T3-L1细胞,以CCK8、EdU试剂盒检测miR-196a-5p对3T3-L1前脂肪细胞增殖的影响作用;(4)运用油红O染色、甘油三酯测定评估miR-196a-5p对3T3-L1细胞分化的影响;(5) RT-PCR检测miR-196a-5p对前脂肪细胞增殖、分化相关基因的影响;(6)结合前人文献,运用生物信息软件、萤光素酶报告系统对miR-196a-5p调控脂肪细胞分化的靶基因进行筛选和验证。结果:(1) miR-196a-5p在肥胖小鼠脂肪组织中高表达,在3T3-L1前脂肪细胞分化过程中先升高后下降;(2)与阴性对照组相比,mimics转染抑制了3T3-L1细胞增殖,inhibitors转染促进了3T3-L1细胞增殖;(3)与阴性对照组相比,mimics组积累了大量油红着色的脂滴,甘油三酯含量增多,而inhibitors组的脂滴少而小,甘油三酯含量相对降低;(4)与阴性对照组相比,mimics转染抑制了增殖标志基因Cyclin D1、Cyclin E、CDK2和CDK4表达,促进了分化标志基因PPARγ、C/EBPα、LPL、aP2等的表达,inhibitors转染则表现出与mimics转染相反的作用;(5) miR-196a-5p可显著抑制野生型MAP4K3和MAPK1 3'UTR萤光素酶活性,而突变绑定位点可废除该抑制效应。结论:miR-196a-5p不仅可抑制3T3-L1前脂肪细胞增殖,还可促进其诱导分化、沉积脂滴;miR-196a-5p可能通过靶向调节MAP4K3和MAPK1来介导3T3-L1前脂肪细胞分化。     

柽柳黄素对3T3-L1脂肪细胞胰岛素抵抗的影响及AMPK信号通路的作用机制

为探讨柽柳黄素对3T3-L1脂肪细胞胰岛素抵抗的影响及AMPK信号通路的作用机制,本研究利用地塞米松诱导3T3-L1脂肪细胞,建立胰岛素抵抗模型,通过给药后检测细胞对Glu的摄取量和细胞内TG的含量,并采用qRT-PCR对AMPK信号通路中相关基因进行检测,利用分子对接软件对AMPK信号通路中相关蛋白进行分子对接,进一步采用Western blot进行蛋白检测。研究结果表明,当柽柳黄素作用48 h后,高低剂量组均显著增加细胞对Glu的摄取(P<0.01),高剂量组显著降低细胞内TG含量(P<0.05);作用机制显示柽柳黄素具有显著提高AMPK(P<0.01)和降低FAS(P<0.05)基因的表达,能与FAS蛋白具有较好的分子对接,可增加P-AMPK、P-ACC、P-PKB和PPARα和抑制FAS蛋白的表达。该研究说明柽柳黄素可增强胰岛素抵抗模型3T3-L1脂肪细胞对Glu的摄取,降低TG在细胞内的含量,其作用机制可能与AMPK信号通路中相关基因和蛋白调节有关。     

胰岛素对βTC-3 细胞胰岛素受体表达的作用

目的:观察外源性胰岛素对小鼠胰岛β细胞瘤细胞株βTC-3细胞胰岛素受体表达的影响。方法:采用免疫荧光细胞化学技术结合激光扫描共聚焦显微镜观察高浓度胰岛素(100 IU/ml)刺激不同时间(0 min、30 min、60 min、120 min、240 min),培养的βTC-3细胞胰岛素受体的表达,用Image pro plus软件对胰岛素受体的荧光强度进行了半定量分析。结果:与0 min比较,胰岛素孵育30 min、60 min、120 min、240 min时胰岛素受体荧光强度均明显下降(P<0.05)。结论:高浓度胰岛素孵育βTC3细胞后,可明显下调胰岛素受体的表达,这可能是高胰岛素血症导致胰岛素抵抗产生的机制之一。     

胰岛素对βTC-3细胞胰岛素受体表达的作用

目的：观察外源性胰岛素对小鼠胰岛β细胞瘤细胞株βTC-3细胞胰岛素受体表达的影响。方法：采用免疫荧光细胞化学技术结合激光扫描共聚焦显微镜观察高浓度胰岛素（100 IU/ml）刺激不同时间（0 min、30 min、60 min、120 min、240 min）,培养的βTC-3细胞胰岛素受体的表达,用Image pro plus软件对胰岛素受体的荧光强度进行了半定量分析。结果：与0 min比较,胰岛素孵育30 min、60 min、120 min、240 min时胰岛素受体荧光强度均明显下降（P〈0.05）。结论：高浓度胰岛素孵育βTC3细胞后,可明显下调胰岛素受体的表达,这可能是高胰岛素血症导致胰岛素抵抗产生的机制之一。     

TLR4 对人骨骼肌细胞炎症因子表达及胰岛素抵抗的影响

目的:研究TLR4对脂多糖(LPS)及Polymymin B(PMB)作用下的人骨骼肌细胞的炎症因子表达的影响及其在细胞胰岛素抵抗中的作用。方法:通过脂多糖(LPS)及Polymymin B(PMB)干预骨骼肌细胞24h,再用胰岛素刺激1h后,Real-time PCR检测检测骨骼肌细胞TLR4、MyD88、TNF-αmRNA的表达;Western blot检测TLR4,Myd88和CRP的表达;葡萄糖氧化酶法(GOD-POD法)检测细胞培养液中葡萄糖浓度。结果:TLR4高表达可以使炎症因子的表达增高,细胞培养液中的葡萄糖浓度增高;TLR4低表达可使炎症因子的表达降低,细胞培养液中的葡萄糖浓度没有明显变化。结论:TLR4调控了炎症因子的表达,继而可以引起胰岛素敏感性的改变,影响了胰岛素抵抗的发生。     

灵芝多糖对AD模型大鼠海马组织Caspase-3和FasL表达的影响

目的研究灵芝多糖（GLP）对阿尔茨海默病（AD）大鼠海马组织半胱氨酰天冬氨酸特异性蛋白静3（caspase-3）和FasL基因以及空间学习记忆能力的影响。方法双侧海马内一次性注射淀粉样多肽25-35片段（Aβ25—35）制作大鼠AD模型,治疗组24h后腹腔注射灵芝多糖水溶液,1周后进行Morris水迷宫检测大鼠空间学习记忆能力变化。采用免疫组织化学法和逆转录-聚合酶链式反应法（RT-PCR）分别检测大鼠海马组织caspase-3蛋白的表达量和FasL基因的表达,以及灵芝多糖对上述各指标的影响。结果灵芝多糖能明显改善AD模型大鼠低下的空间学习记忆能力。显著降低模型大鼠海马组织caspase-3和FasL的表达,而且灵芝多糖能明显改善模型大鼠脑组织海马CAI区神经元的退行性变化。结论灵芝多糖能降低海马组织内FasL和caspase-3表达量,改善海马CAI区神经元的退行性变化。对老年性痴呆大鼠学习记忆能力可能有增强和提高作用。     

Kaempferitrin inhibits GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes

Insulin stimulated GLUT4 (glucose transporter 4) translocation and glucose uptake in muscles and adipocytes is important for the maintenance of blood glucose homeostasis in our body. In this paper, we report the identification of kaempferitrin (kaempferol 3,7-dirhamnoside), a glycosylated flavonoid, as a compound that inhibits insulin stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. In the absence of insulin, we observed that addition of kaempferitrin did not affect GLUT4 translocation or glucose uptake. On the other hand, kaempferitrin acted as an inhibitor of insulin-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes by inhibiting Akt activation. Molecular docking studies using a homology model of GLUT4 showed that kaempferitrin binds directly to GLUT4 at the glucose transportation channel, suggesting the possibility of a competition between kaempferitrin and glucose during the transport. Taken together, our data demonstrates that kaempferitrin inhibits GLUT4 mediated glucose uptake at least by two different mechanisms, one by interfering with the insulin signaling pathway and the other by a possible competition with glucose during the transport.     

Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cells

Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A thin layer of cytosol right adjacent to the plasma membrane was illuminated and successively imaged at 5 Hz under a total internal reflection fluorescence microscope with a penetration depth of 136 nm. Employing single particle tracking, the three-dimensional subpixel displacement of single GSV was tracked at a spatial precision of 22 nm. Both the mean square displacement and the diffusion coefficient were calculated for each vesicle. Tracking results revealed that vesicles moved as if restricted within a cage that has a mean radius of 160 nm, suggesting the presence of some intracellular tethering matrix. By constructing the histogram of the diffusion coefficients of GSVs, we observed a smooth distribution instead of the existence of distinct groups. The result indicates that GSVs are dynamically retained in a continuous and wide range of mobility rather than into separate classes.     

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.     

Essential role of PI-3K, ERKs and calcium signal pathways in nickel-induced VEGF expression

Exposure to a highly nickel-polluted environment has the potential to cause a variety of adverse health effects, such as the respiratory tract cancers. Since numerous studies have demonstrated that nickel generally has weak mutagenic activity, research focus had turned to cell signalling activation leading to gene modulation and epigenetic changes as a plausible mechanism of carcinogenesis. Previous studies have revealed that nickel compounds can induce the expression of vascular endothelial growth factor (VEGF), which is a key mediator of angiogenesis both in physiological and pathologic conditions. In the present study, we investigated the potential roles of PI-3K, ERKs, p38 kinase and calcium signalling in VEGF induction by nickel in Cl 41 cells. Exposure of Cl 41 cells to nickel compounds led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PI-3K inhibitor, wortmannin or Ly294002, resulted in a striking inhibition of VEGF induction by nickel compounds, implicating the role of PI-3K in the induction. However, mTOR, one of downstream molecules of PI-3K, may not contribute to the induction because pre-treatment of Cl 41 cells with its inhibitor, rapamycin, did not show obvious decrease in nickel-induced VEGF expression. Furthermore, pre-treatment of Cl 41 cells with MEK1/2-ERKs pathway inhibitor, PD98059, significantly inhibited VEGF induction by both NiCl₂ and Ni₃S₂, whereas p38 kinase inhibitor, SB202190, did not impair the induction. Pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, inhibited VEGF induction by nickel. Collectively these data demonstrate that PI-3K, ERKs and cytosolic calcium, but not p38 kinase, play essential roles in VEGF induction by nickel compounds. The first two authors have contributed equally to this work.     

PI3K is negatively regulated by PIK3IP1, a novel p110 interacting protein

Signaling initiated by Class Ia phosphatidylinositol-3-kinases (PI3Ks) is essential for cell proliferation and survival. We discovered a novel protein we call PI3K interacting protein 1 (PIK3IP1) that shares homology with the p85 regulatory PI3K subunit. Using a variety of in vitro and cell based assays, we demonstrate that PIK3IP1 directly binds to the p110 catalytic subunit and down modulates PI3K activity. Our studies suggest that PIK3IP1 is a new type of PI3K regulator.     

Characterization of GLUT4-containing vesicles in 3T3-L1 adipocytes by total internal reflection fluorescence microscopy

Insulin-responsive GLUT4 (glucose transporter 4) translocation plays a major role in regulating glucose uptake in adipose tissue and muscle. Whether or not there is a specialized secretory GSV (GLUT4 storage vesicle) pool, and more importantly how GSVs are translocated to the PM (plasma membrane) under insulin stimulation is still under debate. In the present study, we systematically analyzed the dynamics of a large number of single GLUT4-containing vesicles in 3T3-L1 adipocytes by TIRFM (total internal reflection fluorescence microscopy). We found that GLUT4-containing vesicles can be classified into three groups according to their mobility, namely vertical, stable, and lateral GLUT4-containing vesicles. Among these groups, vertical GLUT4-containing vesicles exclude transferrin receptors and move towards the PM specifically in response to insulin stimulation, while stable and lateral GLUT4-containing vesicles contain transferrin receptors and show no insulin responsiveness. These data demonstrate that vertical GLUT4-containing vesicles correspond to specialized secretory GSVs, which approach the PM directly and bypass the constitutive recycling pathway. Contributed equally to this work Supported by the National Natural Science Foundation of China (Grant Nos. 30470448 and 30130230), the National key Basic Research Program of China (Grant No. 2004CB720000), the Knowledge Innovative Program of The Chinese Academy of Sciences (Grant Nos. KSCX2-SW-224 and Y2004018), the Li Foundation and the Sinogerman Scientific Center.     

17β-Estradiol treatment is unable to reproduce p85α redistribution associated with gestational insulin resistance in rats

Maternal metabolic adaptations are essential to ensure proper fetal development. According to changes in insulin sensitivity, pregnancy can be divided into two periods: early pregnancy, characterized by an increase in maternal insulin sensitivity, and late pregnancy, in which there is a significant increase in insulin resistance. The aims of the present work were two-fold: firstly, the molecular mechanisms associated with the development of pregnancy-related insulin resistance in peripheral tissues, mainly retroperitoneal adipose tissue and skeletal muscle, were studied in pregnant rats at 6, 11, and 16 days gestation. Secondly, the role of 17β-estradiol in this process was elucidated in an animal model consisting of ovariectomized rats treated with 17β-estradiol to mimic plasma gestational levels. The results support the conclusion that retroperitoneal adipose tissue plays a pivotal role in the decrease in insulin sensitivity during pregnancy, through a mechanism that involves p85α redistribution to the insulin receptor and impairment of Glut4 translocation to the plasma membrane. Treatment with 17β-estradiol did not reproduce the molecular adaptations that occur during pregnancy, suggesting that other hormonal factors presents in gestation but absent in our experimental model are responsible for p85α redistribution to the insulin receptor.     

ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin activates glucose transport by promoting translocation of the insulin-sensitive fat/muscle-specific glucose transporter GLUT4 from an intracellular storage compartment to the cell surface. Here we report that an optimal insulin effect on glucose uptake in 3T3-L1 adipocytes is dependent upon expression of both PIKfyve, the sole enzyme for PtdIns 3,5-P(2) biosynthesis, and the PIKfyve activator, ArPIKfyve. Small-interfering RNAs that selectively ablated PIKfyve or ArPIKfyve in this cell type depleted the PtdIns 3,5-P(2) pool and reduced insulin-activated glucose uptake to a comparable degree. Combined loss of PIKfyve and ArPIKfyve caused further PtdIns 3,5-P(2) ablation that correlated with greater attenuation in insulin responsiveness. Loss of PIKfyve-ArPIKfyve reduced insulin-stimulated Akt phosphorylation and the cell surface accumulation of GLUT4 or IRAP, but not GLUT1-containing vesicles without affecting overall expression of these proteins. ArPIKfyve and PIKfyve were found to physically associate in 3T3-L1 adipocytes and this was insulin independent. In vitro labeling of membranes isolated from basal or insulin-stimulated 3T3-L1 adipocytes documented substantial insulin-dependent increases of PtdIns 3,5-P(2) production on intracellular membranes. Together, the data demonstrate for the first time a physical association between functionally related PIKfyve and ArPIKfyve in 3T3-L1 adipocytes and indicate that the novel ArPIKfyve-PIKfyve-PtdIns 3,5-P(2) pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport.     

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1 adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.     

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.