Name: glucose uptake measurement and response to insulin stimulation in in vitro cultured human primary myotubes
Uploaded: 2017-06-25T14:00:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes at JoVE.com

作者承认放射生物学服务（里昂 - 萨德医院）的安妮·查里（AnneCharrié）以及Fond National Fis（FNS）的财务支持。

1.细胞培养基和溶液的制备<ol><li> 培养基的制备 <ol><li>通过补充Ham&#39;s F-10培养基与谷氨酰胺（2mM），青霉素/链霉素（最终5μg/ mL），2％胎牛血清（FCS）和2％血清替代物来制备增殖培养基（PM）。 </li><li>通过用谷氨酰胺（2mM），青霉素/链霉素（最终5μg/ mL）和2％FCS补充Dulbecco&#39;s Modified Eagle培养基（DMEM）来制备分化培养基（DM）。 </li></ol></li><li> 葡萄糖摄取溶液的制备 注意:授权人员只能在受限制的区域内处理放射性物质。材料和废物必须按照适当的程序，指导原则和当地立法处理。 <ol><li>为了制备X-Dulbecco的磷酸盐缓冲盐水（X-DPBS），制备含有0.2％（w / v）（终浓度）牛血清白蛋白umin（BSA）。通过0.2μm过滤器过滤溶液。储存于4°C。 </li><li>为了制备冷的2-脱氧-D-葡萄糖（2dG）溶液，称重16.4mg的2dG并溶解在10mL蒸馏水中以获得10mM溶液。储存于4°C。 </li><li>加入600μL冷2dG和6μL放射性标记的[ 3 H] 2dG至5400μLX-DPBS，得到放射性标记的2dG（2dG *）溶液。 注意:最终浓度为1 mM 2dG，标记为1μCi/ mL。 <ol><li>放置放射性标记的2dG *溶液的20μL等分试样（TC20）。 </li></ol></li></ol></li><li> 孵育混合物的制备 <ol><li>对于细胞松弛素B混合物，加入2μL20mM细胞松弛素B至2mL放射性标记的2dG *溶液。 注意:细胞松弛素B的储备溶液在二甲基亚砜（DMSO）中为10mg / mL。 </li><li>对于DMSO混合物，向剩余的4mL放射性标记的2dG *溶液中加入4μLDMSO。 </li></OL&gt; </li></ol> 2.人原代肌细胞培养<ol><li> 用人类肌肉卫星细胞播种6孔板 注意:从冷冻小瓶（含有25万个细胞）内部使用（详见参考资料24 ）或市售的人类肌肉卫星细胞。给出250,000个细胞的以下程序，以获得在单一条件下测量葡萄糖摄取所必需的一个6-孔板。 <ol><li>在预热的水（37℃）中快速解冻冷冻的小瓶内部的24或人类肌肉卫星细胞的商业制剂，直到小瓶中只剩下一小块冰块。 </li><li>直接倒入含有10mL预热（37℃）PM的50mL塑料管中。 </li><li>以500×g离心5分钟，弃去上清液。 </li><li>用18mL预热的PM轻轻重悬细胞沉淀（每个获得42,000个细胞）3 mL培养基）。在6孔板（9.6cm 2 ）的每个孔中分配3mL。 注意:对于以下条件，板的六个单独的孔需要进行葡萄糖摄取的重复测量:被动运输抑制（孔1和2），基础速率（孔3和4）和胰岛素刺激速率（孔5和6）。重复几个六孔板需要不同的处理。 </li><li>在标准培养条件（37℃，5％CO 2 ）中孵育，直到细胞达到90％汇合。 注意:根据细胞批次，此步骤需要48 - 72 h。在此步骤中不要更换介质。 </li></ol></li><li> 肌肉细胞分化 <ol><li>去除PM（48-72小时后），并用预热的DM（3mL /孔）代替。在37℃，5％CO 2下孵育。 注意:分化需要五天才能达到细胞排列和多核的稳定状态。通常，主要肌管在1g / L葡萄糖培养基中培养。因此，为了避免在培养过程中葡萄糖消耗，用3 mL培养基填充平板，以确保足够的葡萄糖底物在任何时间都可用于细胞。 </li><li>每两天更换DM培养基。 注意:从这一点来看，肌管长达7天是稳定的，没有任何显着的变化，并且可以随时进行葡萄糖摄取测量。 </li></ol></li><li> 肌肉细胞治疗（可选） 注意:在胰岛素刺激和葡萄糖摄取测量之前，主要肌管可以治疗数天以诱导修饰（药物测试，信号通路的抑制剂/激活剂等 ）。肌肉细胞可以进行任何可能对胰岛素敏感性影响的治疗，葡萄糖摄入测量将量化这种影响。例如，肌肉细胞与饱和脂肪酸棕榈酸酯的孵育促进胰岛素抵抗，细胞显示减少i苏木精刺激葡萄糖摄取。 <ol><li>准备12 mL补充有10％BSA（不含脂肪酸）和0.5 mL棕榈酸酯（PALM）的DM。准备12 mL补充有10％BSA（不含脂肪酸）的DM。 </li><li>用人原代肌管制备两个6孔板，按2.1和2.2节（分化5天）所述进行培养。 </li><li>在第5天，用2mL PBS洗涤每个孔。向一个平板中加入2mL含有PALM的DM。向其他板中加入2mL仅含有DM的BSA。 </li><li>在37℃，5％CO 2下孵育48小时。 </li></ol></li></ol> 3.胰岛素刺激<ol><li>用2mL PBS洗涤分化的肌肉细胞两次。 </li><li>仔细取出PBS，并用3 mL无FCS的DM孵育3 h（37°C，5％CO 2 ）用于血清消耗。 </li><li>用不含FCS的3mL DM替换所有孔中的培养基。向孔5和6添加100nM胰岛素。 </li><li>孵育人肌管培养1小时（37℃，5％CO 2 ）。 </li></ol>葡萄糖摄取<ol><li>胰岛素刺激1小时后，用X-DPBS洗涤两次（每次洗一毫升）。 </li><li>将1mL细胞松弛素B混合物加入孔1和2，加入1mL DMSO混合物至孔3-6。孵育15分钟（37℃，5％CO 2 ）。孵化结束后，立即将板放在冰上。 </li></ol>细胞裂解<ol><li>用1 mL冰冷的PBS清洗细胞两次。 </li><li>用600μL的50mM NaOH溶解各孔中的细胞。在冰上孵育5分钟，并慢慢地与轨道旋转混合。 注意:如果裂解物太粘稠，最多可用1.5 mL NaOH稀释。 </li><li>使用移液管，重悬和收集细胞裂解液。 </li></ol> 6.放射性标记葡萄糖的测定<ol><li>将每个细胞裂解物400μL放入液体闪烁计数小瓶中。准备一个400的阴性对照小瓶μL50mM NaOH和20μLTC20的阳性对照小瓶（来自步骤1.2.3.1）。 </li><li>向每个小瓶中加入4 mL液体闪烁溶液。关闭盖子并彻底混合每个小瓶（1-2秒）。 </li><li>将每个小瓶插入液体闪烁计数器，并根据制造商的说明测量放射性。每个闪烁瓶记录每分钟的计数（CPM）10分钟。 注意:CPM ="每分钟分解"x"计数效率"。 </li></ol> 7.葡萄糖摄取率<ol><li>使用剩余的裂解物（200μL;来自步骤5.2）来测量蛋白质浓度。使用Bradford 25或等效方法确定每个细胞裂解物的蛋白质浓度。计算每个孔的总蛋白质量（Q）（mg）。 </li><li>要获得TC1（1μL放射性标记的2dG *的值），将TC20的CPM值除以20。 </li><li>对于每个小瓶，计算t他的葡萄糖摄取率如下: <img alt="方程" src="/files/ftp_upload/55743/55743eq1.jpg" /> 注意:R以pmol / mg / min测量。井1-2的平均值为被动运输速率，R p 。井3-4的平均值为基础总运输率，R bt 。孔5-​​6的R平均值给出胰岛素刺激的总运输速率R。 <ol><li>计算基础活动运输率（R ba ）如下: <img alt="方程" src="/files/ftp_upload/55743/55743eq2.jpg" /></li><li>计算胰岛素刺激的活动转运率（R ia ）如下: <img alt="方程" src="/files/ftp_upload/55743/55743eq3.jpg" /> 注意:在胰岛素反应性细胞如肌管中，葡萄糖摄取率通常由三个值表示:R ba ，R ia和胰岛素刺激倍数为R ia / R ba 。 </li></ol></li></ol>

作者没有什么可以披露的。

葡萄糖摄取是测试细胞培养上的活化剂或抑制剂以及它们如何影响葡萄糖使用以及细胞对胰岛素反应的能力的关键生物学测量。已经显示这里描述的方法是快速和可靠的，并已被广泛应用于使用来自健康受试者和/或代谢影响患者6,7,10,12,21,26,27,36,37的主要肌管的许多研究。只有一个6孔板，可以获得总基础运输，基础活动运输和胰岛素刺激主动运输的重复率。这三个值完全描述了体外培养的肌肉细胞的胰岛素敏感性。 <p cla原代肌细胞常规地在胶原包被的平板上培养。当使用像C2C12或L6这样的细胞系时，可以使用未涂覆的板。为了最大限度地提高胰岛素反应，在刺激前必须从肌肉细胞中消耗胰岛素，以使其具有最低的基础状态。在本方案中指出的3小时孵育时间可以根据胰岛素和/或血清饥饿时的细胞稳定性进行修饰。用胰岛素孵育3小时后，用放射性标记的葡萄糖孵育15分钟。与胰岛素孵育1小时的目的是允许任何可用的含GLUT4的囊泡转移到膜上，以获得最大的葡萄糖摄取率。以前的测试表明，较短的孵育时间（15和30分钟）没有达到最大的速率。在放射性标记的2dG的15分钟期间胰岛素刺激的持续性不是强制性的，因为它不影响测量（个人数据）。而且，同样的放射性标记<html>可将2dG混合物加入所有测试孔中。对于细胞裂解，NaOH的体积可以根据细胞特性进行修饰。当使用含有高水平脂肪酸的细胞即非常粘稠的裂解液时，NaOH的体积可以增加到1.5 mL。 该方法使用增强测量灵敏度的放射性标记材料，但要求在受控区域执行协议。材料和废物需要按照当地的安全规范处理。对于非设备实验室，其他比色和荧光定量方法有38种。在非放射性测定中，荧光强度可以在用荧光D-葡萄糖类似物2- [ N- （7-硝基苯并-2-氧杂-1,3-二唑-4-基）氨基] - 2-脱氧-D-葡萄糖（2-NBDG）。有了这个模拟，一些研究小组报道，胰岛素在L6细胞中失去生理作用ass ="xref"&gt; 39，而其他组显示胰岛素仍然增加葡萄糖摄取（在人类原发性肌肉细胞中） 38 。由于GLUT4表达低，与体内条件6,16相比，培养的肌肉细胞中胰岛素刺激的葡萄糖摄取量的定量降低。因此，通过其他方法进一步表征培养的肌肉细胞的胰岛素反应性是重要的。使用免疫检测法测定胰岛素信号通路的关键蛋白（如Akt / PKB和/或GSK3）的磷酸化状态可用于14,21,31。在酶水平下，糖原合成，葡萄糖和/或脂质氧化也可以在有和没有胰岛素14,26,31的条件下进行评估。任何其他细胞<html>理论上可以分析所有细胞是否主动或被动地吸收葡萄糖。胰岛素刺激的孔因此可以被任何其他适当的治疗替代。 使用人类初级肌管意味着肌肉细胞已达到与足够的GLUT4蛋白表达一致的分化状态。如果制剂含有非肌肉细胞或基本上成肌细胞，胰岛素刺激条件下的葡萄糖摄取值与基础状态（R ia vs. R ba ）不会有差异，主要是由于GLUT1转运蛋白占优势。 可以将胰岛素加入到原代肌细胞培养基中以促进和增强肌肉细胞增殖和分化。然而，胰岛素也被证明可以在慢性刺激中诱导胰岛素抵抗40,41 ，class ="xref"&gt; 42。在我们的实验中，人类原发性肌肉细胞仅在培养基变化与血清浓度降低时才适当分化。因此，我们不希望在培养过程中添加外部胰岛素，以在评估胰岛素反应性之前保持细胞免受慢性刺激。最后，该方法需要几个洗涤步骤。因此，必须注意不要将细胞从板上分离，因为它可能与原代细胞发生。

骨骼肌是哺乳动物中最大的葡萄糖沉积物，在很大程度上有助于葡萄糖的体内平衡。这种胰岛素反应性组织是由胰岛素刺激引起的葡萄糖摄取的主要部位1 。 在2型糖尿病中，在包括骨骼肌在内的几种组织中观察到胰岛素抵抗，并导致高于正常血糖浓度。因此，确定该组织及其细胞的胰岛素敏感性的水平是重要的，无论目标是表征受试者的缺陷，还是评估旨在改善其的治疗效率。在人或动物受试者中，评估胰岛素敏感性的金标准技术是高胰岛素血症 - 正义血糖钳夹。由DeFronzo于1979年推出，并自3月 4日修改，然后，该方法允许量化全身and组织胰岛素反应性测量为在胰岛素刺激下灌注的葡萄糖的速率以维持正常的血糖浓度。 也可以使用体外肌肉模型在细胞水平进行胰岛素敏感性检测，并且葡萄糖摄取率的测量仍然是量化细胞对胰岛素刺激的生物反应的有效和可靠的工具5,6,7 。实际上，葡萄糖摄取测量量化胰岛素刺激的细胞生物学反应，从胰岛素与其受体结合到富含GLUT4的囊泡的易位，并包括细胞内信号传导和磷酸化级联8 。 这是人类样本的主要兴趣，因为分化的肌管维持供体表型的许多特征，包括代谢性质在患者9,10,11,12中观察到的病症和病症。肌管显示与骨骼肌13,14的结构，代谢和表型相似性，包括葡萄糖转运蛋白15和细胞胰岛素信号传导机制16的表达 。因此，初级肌管中葡萄糖摄取的测量与表征供体的肌肉表型相关，或调查干预（药物，营养或身体活动）对肌肉细胞胰岛素敏感性的影响。 在进行修改胰岛素敏感性17,18的实验时，培养的肌管上葡萄糖摄取的测量也是可靠的工具。 体外 </em&gt;模型适用于可以改善胰岛素反应性的任何化合物的测试，或可以预防或逆转所获得或诱导的胰岛素抵抗19,20,21,22,23 。 在这里我们描述一个详细的培养和区分人肌管的方案，并测量细胞葡萄糖摄取率。该方法适用于人类肌肉前体细胞的任何来源，无论它们来自实验室内的制备，合作或商业供应商。分别来自小鼠和大鼠来源的永生化肌肉细胞系（如C2C12和L6）也可用于本方案的葡萄糖摄取测定。 我们提供了一个详细的方案来定量使用放射性标记的[ 3 H] 2dG的基础和胰岛素刺激状态的速率。 Ť他使用标记的葡萄糖类似物可以准确测定葡萄糖进入与起始原料减少，这是当与原代细胞一起工作时的常见情况。修饰的葡萄糖分子不能进入代谢途径，因此积累在细胞内，允许通过总细胞放射性进行可靠的定量。实验条件包括使用葡​​萄糖转运抑制剂（细胞松弛素B），并且使用和不使用胰岛素进行测量。该组合允许测定葡萄糖活性进入率，以及计算胰岛素反应指数的倍数变化。该方法在单个孵育时间内呈现一剂胰岛素，但是该方案可以容易地修改用于剂量反应或时间过程实验12 。

<table><tbody><tr><td>Human primary muscle cell</td><td>In house preparation from human skeletal muscle biopsies</td><td>In house preparation from human skeletal muscle biopsies</td><td>If not available, use commercial source</td></tr><tr><td>Human primary muscle cell</td><td>Promocell</td><td>C-12530</td><td>Should be cultured with associated media C23060 and C23061</td></tr><tr><td>6-well plate</td><td>Corning</td><td>356400</td><td>BioCoat Collagen I Multiwell Plates</td></tr><tr><td>Ham&#039;s F10</td><td>Dutscher</td><td>L0145-500</td><td>1 g/L glucose</td></tr><tr><td>Glutamine</td><td>Dutscher</td><td>X0551-100</td><td></td></tr><tr><td>penicilin/streptomycin 100x</td><td>Thermo fisher scientific</td><td>15140122</td><td></td></tr><tr><td>Serum substitute UltroserG</td><td>Pall France</td><td>15950.017</td><td>serum substitute in text</td></tr><tr><td>DMEM low glucose</td><td>Dutscher</td><td>L0064-500</td><td>1 g/L glucose</td></tr><tr><td>Fetal Calf Serum</td><td>Eurobio</td><td>CVFSVF00-01</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Dutscher</td><td>L0625-500</td><td>Contains Mg&lt;sup&gt;2+&lt;/sup&gt; (0.5 mM) and Ca&lt;sup&gt;2+&lt;/sup&gt; (0.9 mM)</td></tr><tr><td>Insulin solution human</td><td>Sigma-Aldrich</td><td>I9278</td><td></td></tr><tr><td>2-deoxy-D-glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D6134</td><td></td></tr><tr><td>Albumin bovine</td><td>euromedex</td><td>04-100-812-E</td><td></td></tr><tr><td>fatty acid-free BSA</td><td>Roche</td><td>10,775,835,001</td><td></td></tr><tr><td>palmitate</td><td>Sigma-Aldrich</td><td>P0500</td><td></td></tr><tr><td>Deoxy-D-glucose, 2-[1,2-3H (N)]</td><td>PerkinElmer</td><td>NET328A001MC</td><td>Specific Activity: 5 - 10 Ci (185-370GBq)/mmol, 1 mCi (37MBq</td></tr><tr><td>Cytochalasin B</td><td>Sigma-Aldrich</td><td>c2743</td><td></td></tr><tr><td>PICO PRIAS VIAL 6 mL</td><td>PerkinElmer</td><td>6000192</td><td></td></tr><tr><td>ultima gold MW CA&amp;nbsp;</td><td>PerkinElmer</td><td>6013159</td><td>scintillation liquid</td></tr><tr><td>b&amp;ecirc;ta counter&amp;nbsp;</td><td>PerkinElmer</td><td>2900TR</td><td></td></tr></tbody></table>

glucose uptake measurement and response to insulin stimulation in in vitro cultured human primary myotubes

骨骼肌是哺乳动物中最大的葡萄糖沉积物，在很大程度上有助于葡萄糖的体内平衡。评估肌肉细胞的胰岛素敏感性与所有研究专门用于探索肌肉葡萄糖代谢和表征代谢改变相关。在肌肉细胞中，葡萄糖转运蛋白4型（GLUT4）蛋白质响应于胰岛素转移到质膜，从而允许葡萄糖大量进入细胞。肌肉细胞通过增加葡萄糖摄取速率来响应胰岛素的能力是量化肌肉细胞对胰岛素敏感性的标准读数之一。人原代肌管是一种合适的体外模型，因为细胞维持供体表型的许多特征，包括胰岛素敏感性。这种体外模型也适用于可能影响胰岛素反应性的任何化合物的测试。分化肌管中葡萄糖摄取率的测量反映胰岛素敏感性。 在这种方法中，人体原代肌细胞在体外培养以获得分化的肌管，并且测量有和没有胰岛素刺激的葡萄糖摄取率。我们提供了一个详细的方案来量化使用放射性标记的[ 3 H] 2-脱氧-D-葡萄糖（[ 3 H] 2dG）的被动和活跃的葡萄糖转运率。提供计算方法来量化活性基础和胰岛素刺激率以及刺激折叠。

在第3天，成肌细胞达到汇合（ 图1A ）。在这个阶段的成肌细胞通常是单核的。培养基改变，第8天分化完成（ 图1B ）（方案第2节）。分化5天后，将肌管对准并且通常多核化。在葡萄糖摄取速率测量之前，将人原代肌管进行棕榈酸酯或仅BSA处理。将细胞与BSA（PALM）或BSA单独（BSA）中的棕榈酸酯0.5mM孵育48小时。进行胰岛素刺激（方案3），测定葡萄糖摄取（方案第4节）。 图2显示在对照条件（BSA）和处理条件（PALM）中的葡萄糖摄取率。在细胞松弛蛋白B孵育的肌管中定量非特异性摄取。可以在BSA和PAL中比较基础和胰岛素刺激的葡萄糖摄取率<html> M条件。胰岛素在BSA状态下显着增加葡萄糖转运（p &lt;0.01）。用PALM处理的肌管中胰岛素刺激的葡萄糖摄取率显着降低（p &lt;0.05）。此外，人胰岛素细胞对胰岛素的反应也可以表示为折叠变化。 图3显示与对照相比，用PALM处理的肌管中胰岛素的反应降低（p &lt;0.05）。 尽管培养的肌管中GLUT4蛋白的表达有限，但是原代肌细胞能够随葡萄糖摄取增加而对胰岛素作出反应（ 图2 ）。观察到的平均倍数变化为〜1.3，类似于具有人肌肉细胞的其他组6,7,10,26 ，&gt; 27。用饱和脂肪酸诱导肌肉细胞中的胰岛素抵抗通常在小鼠，大鼠和人类肌肉细胞中被描述，并且已被证明可以降低治疗肌肉细胞28,29,30,31,32,33,34的葡萄糖摄取。我们进行棕榈酸（0.5mM）孵育48小时，观察到基础和胰岛素刺激的葡萄糖摄取速率降低，胰岛素折叠变化减少，反映胰岛素抵抗状态。本手稿中使用的孵育时间已被选为充分证明对葡萄糖摄取的抑制作用。较短的孵育时间也可以产生胰岛素抵抗和降低葡萄糖摄入31,32,33 </sup&gt; ， 34 。 在体外可以使用较低的生理浓度，以及剂量反应和/或时间过程实验，以及与不饱和脂肪酸如油酸酯29,35的作用的比较。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55743/55743fig1.jpg" /> 图1: 体外原代人肌管。显示了使用光学显微镜获得的2个阶段的人肌管的图像。 （ A ）汇合时成肌细胞培养的第3天。 （ B ）在第8天（分化5天），将肌管对齐。刻度棒= 200μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55743/55743fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图2:BSA和PALM条件下胰岛素刺激对人肌管葡萄糖摄取率的影响。将肌管用BSA单独或0.5mM PALM处理48小时。在中等改变和3小时血清消耗后，在不存在（ - ）或存在（+）胰岛素（1小时，100nM）的情况下测量特异性葡萄糖摄取率（pmol / mg / min）。数据是使用来自四个不同供体的肌管的四次独立实验的平均值±sem。 ＃P &lt;0.05与基础值相比; * P &lt;0.05与BSA胰岛素刺激值相比。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55743/55743fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55743/55743fig3.jpg" /> 图3:折叠IBSA和PALM培养条件下的nsulin反应。刺激胰岛素与BSA和PALM条件下基础葡萄糖转运率的比例。该比例给出胰岛素刺激后的倍数葡萄糖摄取率（100nM）。 * P &lt;0.05与BSA条件（n = 4）相比。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55743/55743fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes at JoVE.com

体外培养的人原代肌管的葡萄糖摄取测量和对胰岛素刺激的反应|生物学 |视频

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

在这种方法中，人原代肌细胞在体外培养以获得分化的肌管，并测量葡萄糖摄取率。我们提供了一个详细的方案，使用放射性标记的[ 3 H] 2-脱氧-D-葡萄糖来量化基础和胰岛素刺激状态的速率。

葡萄糖摄取测量和对胰岛素刺激的反应<em&gt;体外</em&gt;培养的人类初级肌管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

glucose-uptake-measurement-response-to-insulin-stimulation-in-vitro

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Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

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Metabolism

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19.8K 次观看.  University of Lyon. 在这种方法中，人原代肌细胞在体外培养以获得分化的肌管，并测量葡萄糖摄取率。我们提供了一个详细的方案，使用放射性标记的[ 3 H] 2-脱氧-D-葡萄糖来量化基础和胰岛素刺激状态的速率。 

视频: Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

科研

生物化学

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

生物学

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 对高脂饮食喂养小鼠体内葡萄糖代谢的影响研究 (英文)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

医学

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

神经科学

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic &#946; cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

使用共同分泌的Luciferase代理物测量相对胰岛素分泌

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

测量小鼠分离和孵育成熟骨骼肌中胰岛素和收缩刺激的葡萄糖摄取

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

In Vitro Glucose Depletion Assay: A Fluorescence-Based Method to Quantify Cellular Glucose Uptake via Extracellular Glucose Depletion Measurement

Source: Kumar, S. B. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63681">Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues&#160;Ex Vivo.</a>&#160;J. Vis. Exp.&#160;(2022)
This video demonstrates an in vitro assay to indirectly measure intracellular glucose uptake. The assay uses a fluorescently-labeled glucose analog to establish a correlation between extracellular glucose depletion and intracellular glucose uptake.

体外葡萄糖耗竭测定:一种通过细胞外葡萄糖耗竭测量量化细胞葡萄糖摄取的基于荧光的方法

Source: Kumar, S. B. et al., Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues&#160;Ex Vivo.&#160;J. Vis. ...

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Measurement of Extracellular Fluorescent Glucose Analog Depletion: A Surrogate Measurement Technique to Assess Glucose Uptake in Mouse Organs Ex Vivo

Source: Kumar, S. B. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63681">Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo.</a>&#160;J. Vis. Exp.&#160;(2022)
In this video, we demonstrate an ex vivo technique to measure extracellular glucose analog depletion in harvested mouse organs. It involves incubating the mouse tissue in a solution containing fluorescently-labeled deoxyglucose, which is taken up by the cells. The glucose analog uptake can be indirectly determined by measuring the fluorescence of the incubated medium.

葡萄糖摄取测量和对胰岛素刺激的反应

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细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

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此视频中的章节

细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

流式细胞仪定量测定GLUT4转位到质膜

口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 对高脂饮食喂养小鼠体内葡萄糖代谢的影响研究 (英文)

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

使用共同分泌的Luciferase代理物测量相对胰岛素分泌

测量小鼠分离和孵育成熟骨骼肌中胰岛素和收缩刺激的葡萄糖摄取

体外葡萄糖耗竭测定:一种通过细胞外葡萄糖耗竭测量量化细胞葡萄糖摄取的基于荧光的方法

细胞外荧光葡萄糖类似物耗竭的测量:一种评估小鼠离体器官葡萄糖摄取的替代测量技术

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取