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在自觉的,奔放的小鼠正常血糖高胰岛素 - 夹钳

1, 2,3, 3, 2,3, 3, 4, 2,3, 2,3

1Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute at Lake Nona, 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 3Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 4Department of Pediatrics and Cellular and Integrative Physiology, Indiana University School of Medicine

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    Summary

    高胰岛素 - 正常血糖钳,或胰岛素钳夹,是评估胰岛素作用的黄金标准

    Date Published: 11/16/2011, Issue 57; doi: 10.3791/3188

    Cite this Article

    Ayala, J. E., Bracy, D. P., Malabanan, C., James, F. D., Ansari, T., Fueger, P. T., et al. Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice. J. Vis. Exp. (57), e3188, doi:10.3791/3188 (2011).

    Abstract

    2型糖尿病的特点是胰岛素作用缺陷。高胰岛素-正常血糖钳,或胰岛素钳夹,被广泛认为是“金标准”评估体内胰岛素的作用方法。在胰岛素钳夹,高胰岛素血症是通过一个恒定的胰岛素输注。通过可变利率随之而来的葡萄糖输液维持血糖正常。这个变量的葡萄糖输注率(GIR)确定在整个实验的简要间隔通过测量血糖,并相应调整GIR。 GIR是全身胰岛素作用的指示,小鼠具有增强胰岛素的作用,需要更大的GIR。胰岛素钳夹可以纳入管理同位素2 [14 C]脱氧,以评估具体组织对葡萄糖的摄取和[3 - 3 H]葡萄糖评估胰岛素的能力,抑制了内源性葡萄糖的外观(endoRa)的速度,一个标记肝葡萄糖生产,并刺激全身葡萄糖失踪(路)率。

    遗传代谢性疾病的小鼠模型中使用的胰岛素钳夹的小型化导致糖尿病研究的重大进展。执行胰岛素夹具的方法不同实验室之间。重要的是要注意在胰岛素钳夹执行的方式,可以显着影响所取得的成果。我们发表了不同的方法进行全面评估,在自觉小鼠以及四种常用自交系的鼠标,使用各种钳技术2株的代谢反应的评价胰岛素夹具。在这里,我们目前范德比尔特鼠标代谢表型中心(MMPC;网址:www.mc.vanderbilt.edu / MMPC)的开发意识,奔放的小鼠胰岛素夹具上执行协议。这包括在胰岛素钳夹使用的导管植入方法的描述。就业协议范德比尔特MMPC采用了独特的的双导管系统 3 。一个导管插入颈内静脉输液。第二个导管插入颈内动脉,允许提供血液样本,而不需要限制或处理鼠标。这种技术提供了一个显著的优势,为最常用的方法取得的血液样本在胰岛素夹这是样品从切断的尾巴尖。这后一种方法不同,从动脉导管取样是没有压力的鼠标1。我们还描述了使用同位素示踪注入评估组织特异性胰岛素作用的方法。我们还提供适当的胰岛素夹具取得的成果介绍的指引。

    Protocol

    1。导管和鼠标天线抽样访问的制备(MASA TM )

    1. 准备通过动脉导管插入一块6厘米的硅橡胶管图1A所示(0.012英寸内径)1.3厘米的PE - 10片(0.011英寸内径)。斜角用手术刀尖端的PE - 10,因此硅橡胶锥尖底的长度为0.9厘米。
    2. 准备通过滑动的硅橡胶管的1毫米的片(0.020英寸内径)1.1厘米的一块6厘米的硅橡胶管,如图1B所示(0.012英寸内径)的斜面结束静脉导管。 1毫米的硅橡胶一块被用作限制珠。
    3. 要准备一个MASA TM,插入两个3件PE - 20厘米(0.015英寸内径)每两个1.3厘米的25压力表不锈钢接头件。
    4. 安全PE-20/connectors相互滑动一块5毫米的硅橡胶管(内径0.040英寸),超过该地区的钢管和PE - 20。
    5. 钢管的弯曲到〜120 °角,并单独〜45 °每管。
    6. 医疗硅酮胶dollop钻机将完成这样的PE - 20管垂直和超越胶( 图1C)不锈钢管的两端延伸。允许此设置为24小时。

    2。手术导尿

    1. 手术前,用70%乙醇消毒导管,肝素生理盐水填充(肝素200 U / ml生理盐水),并插入不锈钢插头。
    2. 麻醉鼠标,最好使用一个方法,不断提供麻醉剂(如吸入异氟醚)。
    3. 使用无菌技术,去除头发从切口网站使用快船和/或脱毛霜,由优碘擦洗皮肤用酒精消毒。导管插入,删除在头发上从下颌左肋上方的锁骨之间的区域扩大。头部背后的导管外,去除头发颅底interscapular地区之间的区域,由优碘擦洗皮肤用酒精消毒。
    4. 及其对变暖的表面背面放置鼠标和手术显微镜下观赏区。安全与手术磁带的尾巴和四肢。安全提供麻醉在鼻锥头。
    5. 制作一个小的垂直正中切口5毫米头胸骨。使用镊子,生硬的解剖组织揭露左胸锁乳突肌肌。反映这种肌肉暴露左侧颈内动脉。轻轻挑逗从动脉结缔组织。正是在这一点上,重要的隔离从动脉的迷走神经,破坏动脉或神经。
    6. 孤立的动脉,用丝线缝合结扎头侧结束。松散结另一个缝合件暴露的船只的尾部。
    7. 钳与微serrefine钳尾结束的船只和正下方的弹簧剪刀结扎切断。小心地插入导管尽可能钳。小心释放微serrefine钳和推进导管的硅橡胶,PE的交界处。
    8. 把两个连写安全导管和确认,通过导管的自由端连接到采样注射器导管样本。
    9. 设为另一个中线右侧切口5毫米和2毫米左右的尾鳍来的第一个切口。使用镊子,生硬的解剖组织揭露和孤立的右颈内静脉。
    10. 仔细结扎丝线缝合头和松散的领带在另一块缝合尾结束。
    11. 略低于头结扎弹簧剪刀剪下,插入导管的限制珠。领带珠背后的缝合,并确认导管样品。
    12. 涂Rn中的鼠标,使肩胛骨之间的一个小切口。
    13. 隧道14号针头从皮肤切口动脉导管下,鼠标的正面,背面interscapular切口。穿过针的动脉导管,exteriorize在鼠标的背面。颈内静脉导管重复interscapular切口背面前通过隧道从切口部位鼠标右侧的14号针头在皮肤下。
    14. 钳与微serrefine钳在肩胛骨之间的切口部位的动脉导管。切〜1厘米以上钳导管。将MASA TM面向鼠标的头部与不锈钢连接器。动脉导管连接不锈钢连接器,对鼠标的左侧指出。小心,以确保没有孔或导管扭结。重复静脉导管,它连接到不锈钢连接器,指向鼠标的右侧。
    15. MASA TM插入肩胛骨之间的切口。 PE - 20管颈内静脉导管应该是鼠标的右侧和PE - 20油管对应的动脉导管,应当向左侧。
    16. 关闭用尼龙缝合腹侧和背侧切口。缝合背收盘,可通过MASA TM硬化硅运行安全到位。确认通畅导管冲洗液中含有肝素生理盐水和抗生素的使用,以尽量减少感染的风险。立即恢复加热,清洁笼子里放置鼠标在图2显示了成品。
    17. 让鼠标恢复至少5天。显示器的重量和整体健康。利用适当的手术后的镇痛方案,由该机构的动物保健批准和使用委员会。

    3。高胰岛素 - 正常血糖钳

    1. 快速鼠标5 - 6H。作为参考,时间t = 0分钟是指到快结束的胰岛素和葡萄糖输液(即钳期)开始。
    2. 如图3所示为一个典型的实验设置和时间线。使用微Renathane或同等学历的输液管和采样线。暂停鼠标以上的双通道旋转。作为鼠标和输液/采样注射器之间的枢纽。在实验过程中,鼠标仍然在家里笼或类似的容器,是拴在旋转。
    3. 连接鼠标之前,与肝素生理盐水填充的动脉取样线(肝素10 U / ml生理盐水),并将其放置在该行的底端不锈钢接头。保留与肝素生理盐水连接到采样线的顶部(结算注射器)的注射器。这将是用于绘制血桑普LES。
    4. 填写与非肝素化生理盐水静脉滴注从输液的旋转的端口(分部图3A a)所有的方式,该行的底部开始。插线的最高端,并放置一个不锈钢接头(或Y连接,如果丸是要管理)在底行。如果是被注入葡萄糖同位素示踪(如[3 - 3 H]葡萄糖),安全的1 ml注射器,输液注射器含示踪剂。填充用生理盐水代替示踪( 图3B)静脉输液线。
    5. 进入快速3小时,权衡鼠标和颈内静脉和动脉导管输液和采样线连接的PE - 20,分别。
    6. 如果管理[3 - 3 H]葡萄糖示踪剂,开始催芽连续示踪输液在t = -90分钟( 图3C)。一个典型的吸剂量是1μCi。准备一个0.05μCi/μL[3 - 3 H]葡萄糖solution在非肝素化生理盐水。装入1 ml注射器的解决方案和安全注射器,输液泵。管理注入20μL/分钟1分钟吸剂量。按照平衡时间为90分钟的连续输注的0.05μCi/分钟(1μL/分钟)。
    7. 准备胰岛素和葡萄糖infusates。胰岛素是准备在非肝素化生理盐水含3%血浆作为载体(也可以使用适当的BSA浓度)。血糖infusates是在不同浓度(5,20和50%)上市。
    8. 准备获得捐助者的鼠标,最好是一脉相承的背景作为实验小鼠全血生理盐水冲洗erythorocyte infusate。每学习的鼠标通常为1毫升全血需要。离心机分开红细胞的血液。 10 U / ml的肝素生理盐水洗涤红细胞和离心机丢弃的生理盐水。确定红细胞的体积和重悬在同等体积的10 U / ml的heparinized生理盐水。
    9. 每个infusate绘制成1 ml注射器,每个注射器,以确保个人的输液泵。每个注射器连接4路连接器( 图3)
    10. 在t = -15分钟,慢慢进入结算注射器50-100μL血的血液样本。夹具动脉采样线和删除结算注射器。使用手持式血糖仪,血糖读数通过消除动脉采样线的夹子,使血液流入血糖仪带。
    11. 一旦血糖测量,钳动脉采样线,并插入动脉采样线一个钝针头注射器(抽样注射器)。取出钳,并绘制成采样注射器的血液量(见 ) 。夹具动脉采样线和删除采样注射器。结算注射器插入到动脉采样线。画上的柱塞,以消除任何气泡和重新注入50-100微升血液最初绘制。

    注:采样血液量取决于正在执行的分析。例如,分析[3 - 3 H]葡萄糖的浓度需要10μL血浆,血50μL制定。这将产生等离子体,这是足够的分析,加上额外的血浆如果需要20-30μL。激素和其他代谢产物(如胰岛素,游离脂肪酸)的测量需要额外的血液抽样

    1. 分配EDTA涂层微管的采样注射器的血液。离心,收集血浆。置于冰上,直到研究或立即储存在-20 ° C的血浆
    2. 重复步骤3.12 3.10在T = -5分钟。获得额外的血液用于测量基线血浆胰岛素水平(50μL)。测量绘制成肝素或EDTA处理的毛细管血液的基线血细胞比容。测量获得FROM血浆样品在T = -15和-5分钟代表基线值(即空腹)。
    3. 样品在T = -5分钟后,填补血糖,胰岛素和生理盐水洗红细胞输注线多达4路的连接器。连接附加旋转输液端口(或油管连接Y连接,如果注入[3 - 3 H]葡萄糖)管4路的连接器如图3所示。
    4. 开始先注入生理盐水洗涤红细胞。设定输液速度,以取代在研究期间采样(例如,如果超过120分钟的研究抽样共500μL血,输液速度设置为4.2μL/ min的)的血液总量。相比之下其他infusates,红细胞的解决方案是红色的。注入这一解决方案,首先允许任何潜在的阻力或障碍物识别和纠正在输液线。
    5. 一旦红细胞infusate达到鼠标,开始胰岛素和葡萄糖输液。这是现在T = 0分钟。胰岛素是注入在一个恒定的,预先确定的利率。 •公斤-1•-1的4亩的胰岛素输注率通常会抑制80-100%的内源性葡萄糖生产和刺激葡萄糖失踪2-3倍。初始葡萄糖输注率(GIR)是基于基线血糖水平和以往的经验估计。
    6. 如果注入[3 - 3 H]葡萄糖,可以选择增加示踪输液速度相匹配的葡萄糖营业额估计增加(一般为2-3倍) 。
    7. 率很高的葡萄糖在小鼠的营业额,应取得的血液样本从动脉行不超过每10分钟的血糖浓度在实验期间测量。调整GIR以实现和维持目标血糖正常( 图3C)。这一目标可以有所不同,根据模型或研究的目的。一个良好的目标GLucose浓度为150毫克•DL - 1,因为这是一个典型的6H为议员喂养的C57BL/6J小鼠禁食血糖水平。
    8. 我们的目标是迅速地实现血糖正常,最好是第40-50分钟内,并有葡萄糖和GIR稳定稳态期间(T = 80分钟)开始。
    9. 如果注入[3 - 3 H]葡萄糖,在T = 80,90,100,110和120分钟测量血浆中获得额外的血液[3 - 3 H]葡萄糖具体活动。
    10. 收集更多的血液在t = 100和120分钟测量血浆胰岛素和其他任何激素(S)或代谢物(S)。在T = 110分钟,绘制成肝素或EDTA处理钳测量血细胞比容毛细管血液。
    11. 后的样品在t = 120分钟,2 [14]脱氧可用于具体组织对葡萄糖的摄取量测管理。管理12μCi丸丸线连接到颈采样线( 图3B
    12. 获得血液样本(50μL)T = 2,15,25和35分钟后血浆2 [14C]脱氧葡萄糖水平的测量丸管理的动脉取样线。
    13. 最后一个样本后,用戊巴比妥麻醉输液直接进入动脉行鼠标。快速剖析评估对葡萄糖的摄取(如不同类型,脂肪组织,心脏,大脑的骨骼肌)和任何其他组织(如肝,脾,肾)所需的任何组织。冻结组织在液氮和存储卡在-80℃直至分析。组织对葡萄糖的分析是通过测量2磷酸化的积累[14 C]脱氧葡萄糖在冰冻组织和2 [14C]脱氧从血浆消失。

    4。代表性的成果

    从胰岛素钳夹实验获得的结果的一个例子是如图4所示。这个例子表明,高脂肪的饮食沉淀在小鼠体内的胰岛素抵抗的能力。胰岛素钳夹结果的报告必须包括以下解释:血糖水平的时间当然,日久GIR和血浆胰岛素水平(基线和钳位)。如上所示,空腹血糖( 图4A)和胰岛素( 图4C)水平较高的小鼠喂食高脂肪食物,胰岛素抵抗的指标。呈现出整个钳研究时间的血糖水平( 图4A)课程,让读者评估如何是保持血糖正常,这是钳质量的指标。同样,GIR( 图4B)的时间历程,使读者确定如何迅速达到稳态。这些数据显示,随着时间的课程是显著超过常规的做法在小鼠胰岛素钳文学呈现出2小时的实验信息作为一个单一的基准点,代表从一个未定义的“钳” 期间(4-13)的平均值。在目前的例子中,血糖水平控制和高脂肪喂养的群体之间的平等,但GIR显着降低高脂肪喂养组( 4B )。这是在整个人体胰岛素的作用减值指示。钳胰岛素水平也较高,在高脂肪喂养组( 图4C),进一步支持在这些小鼠的胰岛素耐药表型的存在。使用同位素示踪注入允许评估在特定的组织对胰岛素的作用。 [3 - 3 H]葡萄糖是用来估算的内源性葡萄糖的外观(endoRa),这是一个肝糖生产(HGP)和全身葡萄糖失踪(路)率指数的速度。而胰岛素完全抑制对照组小鼠的人类基因组计划,这是受损的老鼠喂食高脂肪食物(图 4D) 。同样,在能力胰岛素刺激对照组小鼠的路是损害小鼠喂食高脂肪食物(图 4E) 。 2 [14C]脱氧是用来评估糖代谢指数(RG),具体组织对葡萄糖的摄取措施。在这个例子中可以看出,胰岛素刺激骨骼肌葡萄糖摄取受损的老鼠喂食高脂肪食物(图4F)

    图1
    图1:动脉(A)和(二)静脉导管和(C)MASA TM准备。动脉导管插入一个6厘米的片0.012“编号硅橡胶1.3厘米一块约3毫米的PE - 10准备。PE - 10针尖斜面,这样的硅橡胶锥的长度为0.9厘米。静脉导管是由滑动0.020编号硅橡胶“编号硅橡胶1.1厘米为0.012 6厘米的一块斜面”的小片0.020“ID限制珠本身硅橡胶件的行为治疗颈内静脉导管。 MASA TM,每两个1.3厘米的组装25压力表连接器插入到每个PE - 20厘米的两个3件。这些由0.040“编号硅橡胶小块一起举行的接头弯曲角度120 °和45 °角分开,整个大会是沉浸在医疗硅酮胶。

    图2
    图2:插管鼠标。在左侧颈总动脉和右颈内静脉导管手术植入。外部导管的自由端头后面,并连接到一个MASA TM 。 MASA TM插入肩胛骨之间的皮下。这使得在胰岛素钳夹实验的血管通路,无需克制,处理或麻醉鼠标。

    JPG“/>
    图3:胰岛素钳夹实验的设置和时间线的写照。鼠标是拴在一个双通道旋转,作为输液和采样注射器枢纽。不使用示踪剂注入实验的典型设置(A)和同时使用[3 - 3 H]葡萄糖和2 [14 C]脱氧葡萄糖(二)所示。也显示一个程序,建立和执行的胰岛素钳夹(三)时间线。在钳,血液样本( 血 )采取每10分钟测量血糖。 GIR是相应调整,以保持血糖正常。样品葡萄糖为基线血糖,血浆胰岛素,血浆[3 - 3]在T = -15和-5分钟。在T = 80,90,100,110和120和钳位在T = 100和120 min胰岛素样品葡萄糖钳等离子[3 - 3]。 2 [14C]脱氧后的样品在T = 120分钟和B管理lood收集T = 2,15,25和35分钟后。组织后采取的T = 35分钟样品。

    图4
    图4:在控制饮食的小鼠(周)比较高脂肪食物(HFD)对小鼠胰岛素钳夹实验结果。时间动脉血糖(A)和GIR(二),基线和钳胰岛素(C)EndoRa(D)和路(E)和骨骼肌(腓肠肌和股外侧)RG(F)的过程中显示。所有的结果表明,高脂肪喂养的效果,诱导胰岛素抵抗。

    Discussion

    高胰岛素-正常血糖钳,或胰岛素钳夹,被广泛认为是“金标准”评估体内胰岛素的作用方法。这项技术已被应用到几个物种,包括人类,狗,大鼠和小鼠。鉴于转基因小鼠模型的代谢紊乱的数量越来越多,在鼠标中使用的小型化技术的代谢研究提供了显著的进步。

    虽然胰岛素钳夹背后的概念很简单,在实践中有不同的方法执行胰岛素钳夹实验。这不是一个简单的点,因为在实验进行的方式影响结果获得 1 。在这里,我们目前的范德比尔特MMPC使用的协议。和他人之间的协议的主要区别是,我们取得的血液样本中使用的动脉导管。这是在OBT的更广泛使用的方法癌宁切断尖尾4,7,11,12,14-17血液样本。从动脉导管的优点是采样,实验是在有意识地和奔放的鼠标进行。从尾部取样,往往需要克制,并增加了压力指数时,大量的血液样本收购 1 。应激激素刺激内源性葡萄糖生产,损害葡萄糖处置18,19,潜在的胰岛素耐药表型的外观。采样从被切断的尾巴,可能需要特殊的体制动物保护,因为它的压力性质和使用委员会的批准。动脉导管程序的开发,以避免应力撞断了尾巴的鼠标。

    执行胰岛素夹具的一个关键方面的能力,以维持血糖正常。有没有算法能够正确预测血糖读数的基础上,GIR应如何进行调整。手术一样,经验只有通过保持合理的正常血糖进行胰岛素钳夹实验人员将成为精通。重要的是要注意,因为其较高的代谢率从小鼠研究获得的数据将被固有的嘈杂。这使得完整的数据,包括课程的葡萄糖和GIR和血浆胰岛素,endoRa,路和Rg任何读者解释结果的能力是至关重要的绝对值介绍,。在鼠标(比在人类的速度高出约5倍),高糖通量率保证高频率的葡萄糖采样。虽然鼠标的血液量是有限的,每10分钟一次的最低采样频率是必要的,已经取得了足够的钳。

    如图4所示,钳胰岛素水平可以不同群体之间。如饮食干预,转基因操作或背景株的差异等因素,可以影响FAS汀胰岛素水平,继而影响钳胰岛素水平。可问题的解释结果时,钳胰岛素水平是不同的。这可以通过实验处理进行试点实验,选择胰岛素输注率达到相当于钳群体之间的胰岛素水平。另外,生长抑素可以用来抑制胰腺激素的分泌,而胰岛素和胰高血糖素可在实验控制率所取代。后一种方法是比较常用的大鼠比小鼠胰岛素夹。如果不采取这些实验方法,稳态GIR可归钳胰岛素水平,胰岛素敏感指数( )可以从钳数据得出S I = / GIR(G•ΔI),其中G是稳态血糖浓度和ΔI是空腹和钳胰岛素浓度之间的差异。两种方法的一个假设是,钳胰岛素水平达到对胰岛素的敏感性是线性相关的胰岛素水平,根据本集团正在研究的范围内。后者的假设可能不适用于比较时,胰岛素抵抗和胰岛素敏感的群体。理想的情况下,产生胰岛素的剂量反应曲线应选择适当的胰岛素输注。然而,由于更多的实验要求,这是很少这样做。

    动脉插管提供的多功能性,延伸到超出正常血糖夹具的实验方法。例如,高血糖的夹子,其中的葡萄糖注入可变利率维持空腹血糖高血糖相对,可用于评估内源性胰腺 功能在体内2,20 ,21。第一时相胰岛素分泌的测量测试过程中需要频繁采集血液样本(即每2-5分钟),获得尾尖样本时,这是不可行的的。此外,从尾部取样造成的升高儿茶酚胺能削弱胰岛素的分泌和增强胰高血糖素的分泌 22 。胰岛素钳夹协议,也可以进行修改,以使血糖水平下降到相对低血糖评估反监管的反应2,23, 24。动脉插管,也可用于评估在行使25-30糖代谢的动态。在单一时间点进行的常规方法,这是明显的优势超过前和运动后或在孤立肌肉体外。这里介绍的技术也可以用来评估不只是葡萄糖,而且脂肪酸的代谢31。

    Disclosures

    没有利益冲突的声明。

    Acknowledgements

    这项工作得到了批准5 - U24 - DK059637 - 10范德比尔特鼠标代谢表型中心。

    References

    1. Ayala, J. E., Bracy, D. P., McGuinness, O. P., Wasserman, D. H. Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes. 55, 390-397 (2006).
    2. Berglund, E. D., Li, C. Y., Poffenberger, G., Ayala, J. E., Fueger, P. T., Willis, S. E., Jewell, M. M., Powers, A. C., Wasserman, D. H. Glucose metabolism in vivo in four commonly used inbred mouse strains. Diabetes. 57, 1790-1799 (2008).
    3. Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., Magnuson, M. A. Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism. J. Biol. Chem. 272, 22570-22575 (1997).
    4. Kim, H. J., Higashimori, T., Park, S. Y., Choi, H., Dong, J., Kim, Y. J., Noh, H. L., Cho, Y. R., Cline, G., Kim, Y. B., Kim, J. K. Differential effects of interleukin-6 and -10 on skeletal muscle and liver insulin action in vivo. Diabetes. 53, 1060-1067 (2004).
    5. Kim, J. K., Fillmore, J. J., Chen, Y., Yu, C., Moore, I. K., Pypaert, M., Lutz, E. P., Kako, Y., Velez-Carrasco, W., Goldberg, I. J., Breslow, J. L., Shulman, G. I. Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance. Proc. Natl. Acad. Sci. U. S. A. 98, 7522-7527 (2001).
    6. Kim, J. K., Fillmore, J. J., Gavrilova, O., Chao, L., Higashimori, T., Choi, H., Kim, H. J., Yu, C., Chen, Y., Qu, X., Haluzik, M., Reitman, M. L., Shulman, G. I. Differential effects of rosiglitazone on skeletal muscle and liver insulin resistance in A-ZIP/F-1 fatless mice. Diabetes. 52, 1311-1318 (2003).
    7. Kim, J. K., Fillmore, J. J., Sunshine, M. J., Albrecht, B., Higashimori, T., Kim, D. W., Liu, Z. X., Soos, T. J., Cline, G. W., O'Brien, W. R., Littman, D. R., Shulman, G. I. PKC-theta knockout mice are protected from fat-induced insulin resistance. J. Clin. Invest. 114, 823-827 (2004).
    8. Kim, J. K., Gavrilova, O., Chen, Y., Reitman, M. L., Shulman, G. I. Mechanism of insulin resistance in A-ZIP/F-1 fatless mice. J. Biol. Chem. 275, 8456-8460 (2000).
    9. Kim, J. K., Gimeno, R. E., Higashimori, T., Kim, H. J., Choi, H., Punreddy, S., Mozell, R. L., Tan, G., Stricker-Krongrad, A., Hirsch, Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle. J. Clin. Invest. 113, 756-763 (2004).
    10. Kim, J. K., Kim, H. J., Park, S. Y., Cederberg, A., Westergren, R., Nilsson, D., Higashimori, T., Cho, Y. R., Liu, Z. X., Dong, J., Cline, G. W., Enerback, S., Shulman, G. I. Adipocyte-specific overexpression of FOXC2 prevents diet-induced increases in intramuscular fatty acyl CoA and insulin resistance. Diabetes. 54, 1657-1663 (2005).
    11. Kim, J. K., Kim, Y. J., Fillmore, J. J., Chen, Y., Moore, I., Lee, J., Yuan, M., Li, Z. W., Karin, M., Perret, P., Shoelson, S. E., Shulman, G. I. Prevention of fat-induced insulin resistance by salicylate. J. Clin. Invest. 108, 437-446 (2001).
    12. Kim, J. K., Michael, P. revis, Peroni, S. F., Mauvais-Jarvis, O. D., Neschen, F., Kahn, S., Kahn, B. B., R, C., Shulman, G. I. Redistribution of substrates to adipose tissue promotes obesity in mice with selective insulin resistance in muscle. J. Clin. Invest. 105, 1791-1797 (2000).
    13. Kim, J. K., Zisman, A., Fillmore, J. J., Peroni, O. D., Kotani, K., Perret, P., Zong, H., Dong, J., Kahn, C. R., Kahn, B. B., Shulman, G. I. Glucose toxicity and the development of diabetes in mice with muscle-specific inactivation of GLUT4. J. Clin. Invest. 108, 153-160 (2001).
    14. Haluzik, M., Gavrilova, O., LeRoith, D. Peroxisome proliferator-activated receptor-alpha deficiency does not alter insulin sensitivity in mice maintained on regular or high-fat diet: hyperinsulinemic-euglycemic clamp studies. Endocrinology. 145, 1662-1667 (2004).
    15. Haluzik, M., Yakar, S., Gavrilova, O., Setser, J., Boisclair, Y., LeRoith, D. Insulin resistance in the liver-specific IGF-1 gene-deleted mouse is abrogated by deletion of the acid-labile subunit of the IGF-binding protein-3 complex: relative roles of growth hormone and IGF-1 in insulin resistance. Diabetes. 52, 2483-2489 (2003).
    16. Haluzik, M. M., Lacinova, Z., Dolinkova, M., Haluzikova, D., Housa, D., Horinek, A., Vernerova, Z., Kumstyrova, T., Haluzik, M. Improvement of insulin sensitivity after peroxisome proliferator-activated receptor-alpha agonist treatment is accompanied by paradoxical increase of circulating resistin levels. Endocrinology. 147, 4517-4524 (2006).
    17. Kim, H., Haluzik, M., Asghar, Z., Yau, D., Joseph, J. W., Fernandez, A. M., Reitman, M. L., Yakar, S., Stannard, B., Heron-Milhavet, L., Wheeler, M. B., LeRoith, D. Peroxisome proliferator-activated receptor-alpha agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis. Diabetes. 52, 1770-1778 (2003).
    18. Deibert, D. C., DeFronzo, R. A. Epinephrine-induced insulin resistance in man. J. Clin. Invest. 65, 717-721 (1980).
    19. Rizza, R. A., Cryer, P. E., Haymond, M. W., Gerich, J. E. Adrenergic mechanisms for the effects of epinephrine on glucose production and clearance in man. J. Clin. Invest. 65, 682-689 (1980).
    20. Nunemaker, C. S., Wasserman, D. H., McGuinness, O. P., Sweet, I. R., Teague, J. C., Satin, L. S. Insulin secretion in the conscious mouse is biphasic and pulsatile. Am. J. Physiol. Endocrinol. Metab. 290, 523-529 (2006).
    21. Nunemaker, C. S., Zhang, M., Wasserman, D. H., McGuinness, O. P., Powers, A. C., Bertram, R., Sherman, A., Satin, L. S. Individual mice can be distinguished by the period of their islet calcium oscillations: is there an intrinsic islet period that is imprinted in vivo. Diabetes. 54, 3517-3522 (2005).
    22. Halter, J. B., Beard, J. C., Jr, P. orte, D, Islet function and stress hyperglycemia: plasma glucose and epinephrine interaction. Am. J. Physiol. 247, 47-52 (1984).
    23. Jacobson, L., Ansari, T., McGuinness, O. P. Counterregulatory deficits occur within 24 h of a single hypoglycemic episode in conscious, unrestrained, chronically cannulated mice. Am. J. Physiol. Endocrinol. Metab. 290, 678-684 (2006).
    24. Jacobson, L., Ansari, T., Potts, J., McGuinness, O. P. Glucocorticoid-deficient corticotropin-releasing hormone knockout mice maintain glucose requirements but not autonomic responses during repeated hypoglycemia. Am. J. Physiol. Endocrinol. Metab. 291, 15-22 (2006).
    25. Ayala, J. E., Bracy, D. P., James, F. D., Julien, B. M., Wasserman, D. H., Drucker, D. J. The glucagon-like peptide-1 receptor regulates endogenous glucose production and muscle glucose uptake independent of its incretin action. Endocrinology. 150, 1155-1164 (2009).
    26. Fueger, P. T., Bracy, D. P., Malabanan, C. M., Pencek, R. R., Granner, D. K., Wasserman, D. H. Hexokinase II overexpression improves exercise-stimulated but not insulin-stimulated muscle glucose uptake in high-fat-fed C57BL/6J mice. Diabetes. 53, 306-314 (2004).
    27. Fueger, P. T., Bracy, D. P., Malabanan, C. M., Pencek, R. R., Wasserman, D. H. Distributed control of glucose uptake by working muscles of conscious mice: roles of transport and phosphorylation. Am. J. Physiol. Endocrinol. Metab. 286, 77-84 (2004).
    28. Fueger, P. T., Heikkinen, S., Bracy, D. P., Malabanan, C. M., Pencek, R. R., Laakso, M., Wasserman, D. H. Hexokinase II partial knockout impairs exercise-stimulated glucose uptake in oxidative muscles of mice. Am. J. Physiol. Endocrinol. Metab. 285, 958-963 (2003).
    29. Fueger, P. T., Hess, H. S., Posey, K. A., Bracy, D. P., Pencek, R. R., Charron, M. J., Wasserman, D. H. Control of exercise-stimulated muscle glucose uptake by GLUT4 is dependent on glucose phosphorylation capacity in the conscious mouse. J. Biol. Chem. (2004).
    30. Fueger, P. T., Li, C. Y., Ayala, J. E., Shearer, J., Bracy, D. P., Charron, M. J., Rottman, J. N., Wasserman, D. H. Glucose kinetics and exercise tolerance in mice lacking the GLUT4 glucose transporter. J. Physiol. 582, 801-812 (2007).
    31. Shearer, J., Coenen, K. R., Pencek, R. R., Swift, L. L., Wasserman, D. H., Rottman, J. N. Long chain fatty acid uptake in vivo: comparison of [125I]-BMIPP and [3H]-bromopalmitate. Lipids. 43, 703-711 (2008).

    Comments

    21 Comments

    Dear auther
    I would like to know if the disc accomodating the arterial and venus cathters tunneled under the skin and allowing the catheter antenas to protude is a commercial item and if not how do you produce it ?
    Thank you Rona
    Reply

    Posted by: Rona S.November 29, 2012, 8:09 AM

    Dear Rona,
    If you are referring to the MASA, please refer to section 1 of this article - "Preparation of catheters and the Mouse Antenna for Sampling Access". This section describes how to make the disc. Bend two 1.3 cm pieces of ²5 gauge steel tubing to about 1²0 degrees. Insert a 3 cm piece of PE-²0 into each steel tube. Secure both pieces of steel tubing to each other with a 5mm piece of silastic tubing. Dip the steel tubes into a drop of medical grade silicone adhesive such that the PE-²0 is vertical and the free steel tube ends come out of the bottom of the drop at about a 45 degree angle separation. Let the adhesive become solid overnight. I hope this helps.
    Best regards,
    Julio Ayala
    Reply

    Posted by: Julio A.November 29, 2012, 8:46 AM

    Dear Julio
    Yes this was my question
    Thank you for the explenation
    All the best Rona
    Reply

    Posted by: Rona S.November 29, 2012, 9:39 AM

    Hello Julio, I wonder how you maintain catheters. Should I flush them with heparinized saline everyday? Even if flushing everyday, catheters are clogged easily. Do I need higher heparin than 200 U/ml for flushing? Give me some advice, please? Thank you,
    Reply

    Posted by: Teayoun K.March 26, 2014, 1:17 PM

    Hello Teayoun,
    This may depend on the material that you use for the tubing that is attached to the MASA. Originally, we used microrenathane tubing and would flush the lines daily with 200 U/ml hep saline. We have since started using PE-20 instead of microrenathane and find that we do not need to flush the lines (sometimes we will flush the lines 3 days after surgery just for good measure). With either approach, you will inevitably have some catheters clog, but this should not occur often. If you are flushing your lines daily and most of your catheters are still clogging, then there is likely another issue (perhaps the placement of the catheter is not correct?). Is this the arterial or the venous catheter? If it is the arterial catheter, is it sampling on the day of the surgery? How long until it clogs?

    Julio
    Reply

    Posted by: Julio A.March 26, 2014, 2:06 PM

    Julio,

    Thank you for quick response. Yes, I use microrenathane to make venous catheter body, but the tip of catheter is PE-20. If the material itself is problem, I will try PE-20 to make the body part of catheter. The catheter was working well (infusion was easy and smooth as a confirmation after implantation surgery done). Arterial catheter is a commercial one from Alzet (designed for mouse jugular vein, but works well for mouse carotid artery). I've heard some comments from others. heparin causes lipolysis and changing body metabolism and streptokinase may be a problem due to microbial material causing immune response. I'm still not 100% comfortable on catheter maintenance. Metal plug was used, but it's same. I don't know exact reason yet. I wish I could figure it out soon. Thank you,
    Reply

    Posted by: Teayoun K.March 28, 2014, 12:20 PM

    Teayoun,
    I would not recommend using PE or microrenathane for the catheter body (either vein or artery). PE is too rigid and microrenathane is too porous and prone to react (clot). It is my understanding that the Alzet catheters are made with polyurethane which, like the microrenathane, is too porous. I woudl recommend making the catheters as shown in this article (silastic for the vein and silastic with a PE-10 tip for the artery). Heparin will have an effect on lipolysis, but the amounts and frequency with which you flush should not have significant effects on metabolism. Also, when checking the catheters after the surgery, make sure that you can draw blood, not just infuse smoothly. Sometimes you can infuse very smoothly but cannot draw blood, which means that the catheter is not in place.
    Reply

    Posted by: Julio A.March 28, 2014, 3:42 PM

    Dear author,
    Can you please provide a product number and purchasing source for the "medical silicone adhesive" used for MASA construction?
    thank you
    Joe C.
    Reply

    Posted by: Joseph C.April 8, 2014, 5:11 PM

    Hi Joe C.

    You can get it from Fisher:

    FACTOR II INC MEDICAL ADHESIVE SIL TYPE A Catalog No.: NC9938868 Medical Adhesive, Type A

    Good luck!
    Julio
    Reply

    Posted by: Julio A.April 11, 2014, 4:15 PM

    Dear Dr.Ayala,

    We performed surgery following your protocol, 5 days later, animals gain body weight back and both catheters stay open.We try to clamp the blood glucose level around 150mg/dl, The insulin dose is 4mU/kg.min. The fasting glucose level of our mice is around 120, During the H3 tracer infusion phase, the blood glucose level normally dropped to around 100, but will not fluctuate a lot. However when we stared insulin and glucose infusion, the blood glucose can be stable for 40 mins then suddenly drop to below 100. Then we increased GIR, the blood glucose will come back to 150s but will not stay there, after 30 mins to 40 mins it will dip again.
    We dissect the mice after surgery, to make sure there was no leakage.
    We paid attention to small blood sample size,donor blood,and we controlled temperature, we kept environment quiet. We fast mice 3hrs before clamp. Could you please give us some suggestions for trouble shooting? Thanks a lot.
    Reply

    Posted by: Kai M.April 15, 2014, 11:17 AM

    Hello,
    Without seeing our data, particularly the time course of your glucose infusion and glucose levels, I really cannot tell you whether your experience is unusual. When you say that "it dips again", does it dip below 100 mg/dL? Does it dip by 5, 10 mg/dL? How much are you changing the glucose infusion rate? Are these wild-type mice or a transgenic line? You can contact me directly at jayala@sanfordburnham.org if you want to discuss further.
    Reply

    Posted by: Julio A.April 16, 2014, 10:32 AM

    Dear Dr.Ayala,

    I used to use micro-renathane tubing to make venous catheter tip, however Micro-renathane is rigid, half of my catheter penetrate the venous wall, I end up with all my infusate leaked into chest cavity. Then I start to follow your instruction to make venous catheter with silastic tubing. However, I found quite a few my surgery failed because my ligation on the catheter will leak after 3,4 days. No matter how tight I made the ties, or add 1 or two more sutures, it didn't improve. I always found a big lump of infusate formed after clamp subcutaneously near the neck. I am using 6-o silk suture now. Can you please give me some hint to improve the procedure?

    Thanks
    Reply

    Posted by: Kai M.October 2, 2014, 12:32 PM

    Hello Kai. I am Carlo and the hands doing the surgical procedure parts of the video are attached to my body :-). Kidding aside, do the opposite. Try tying over the vessel/catheter with the least possible pressure. The guide is: once you see that the suture knot is deforming the silastic tubing, you are tying too tight! You only need the suture to be snug and it should hold in the vessel without affecting the patency or causing it to leak. Let me know if this tip works.
    Reply

    Posted by: Carlo M.October 2, 2014, 1:29 PM

    Thanks. I will try it!

    Kai
    Reply

    Posted by: Kai M.October 2, 2014, 4:11 PM

    Dear Kai,
    Are you making the silastic "collar" using 0.020" ID silastic as shown in Fig. 1B? If you tie your suture behind this collar, it should secure the catheter in the vessel.
    Reply

    Posted by: Julio A.October 2, 2014, 1:05 PM

    Dr.Ayala, Thanks for your reply, Yes I do, the leakage always happens around the ties before the docking beads. The ties can secure the catheter for about 4days. I doubt if my 7-o suture is too sharp, ( I typo a 6-O suture in the previous comment).so the it damage the wall of blood vessel . Can you tell me what size do you use? Another concern is if the silastic tubing is too soft. The leakage rarely happens when I use Micro-renathane tubing. Thanks.
    Reply

    Posted by: Kai M.October 2, 2014, 1:25 PM

    Also, just out of curiosity, are you making your venous catheter as a single 6cm long piece of silastic? I ask because in your original comment, you referred to the "venous catheter tip". I was just wondering if you were trying to make it the same as the arterial catheter. It should just be a single piece of silastic with the collar slipped over it. I just saw that Carlo Malabanan posted a reply as well. He is an expert at this surgery.
    Reply

    Posted by: Julio A.October 2, 2014, 1:38 PM

    We use 7-0 sutures and silastic tubing and have not had leaking problems with the silastic. I will confer with my colleagues at Vanderbilt to see if they have seen this before.
    Reply

    Posted by: Julio A.October 2, 2014, 1:31 PM

    Dear Malabanan

    Could you kindly explain why 7-o is better than 6?

    Thanks
    Reply

    Posted by: Kai M.October 2, 2014, 2:56 PM

    Dr.Ayala,

    I called the part in fornt of docking beads the "tip" , yes I used a whole piece of silastic tubing as my catheter.Thanks.
    Reply

    Posted by: Kai M.October 2, 2014, 1:47 PM

    Dr.Ayala and Dr.Malabanan
    I figured out the reason for venous leakage is didn't insert the catheter deep enough, since I worry about the tip of venous catheter penetrating the venous wall, I only use 9mm long tip. Which cause a dead space in the vein where blood will stay and form clot, once the clot block the vein, the pressure will build up and eventually burst the venous wall when I start to infuse.Now as you suggested I am using 11mm long tip, which will stay close enough to the heart, and no clot will form before the opening of catheter. Thanks For your help.
    Reply

    Posted by: Kai M.November 4, 2014, 1:04 PM

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