Ayala Julio E, Bracy Deanna P, Malabanan Carlo, James Freyja D, Ansari Tasneem, Fueger Patrick T, McGuinness Owen P, Wasserman David H
Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute at Lake Nona, USA.
J Vis Exp. 2011 Nov 16(57):3188. doi: 10.3791/3188.
Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[(14)C]deoxyglucose to assess tissue-specific glucose uptake and [3-(3)H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd). The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice(1) as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques(2). Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system(3). One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse(1). We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.
2型糖尿病的特征是胰岛素作用存在缺陷。高胰岛素-正常血糖钳夹技术，即胰岛素钳夹技术，被广泛认为是评估体内胰岛素作用的“金标准”方法。在胰岛素钳夹实验中，通过持续输注胰岛素实现高胰岛素血症。通过以可变速率同时输注葡萄糖来维持血糖正常。这种可变的葡萄糖输注速率（GIR）是通过在整个实验过程中每隔一段时间测量血糖并相应调整GIR来确定的。GIR可反映全身胰岛素作用，因为胰岛素作用增强的小鼠需要更高的GIR。胰岛素钳夹技术可结合给予同位素2-[(14)C]脱氧葡萄糖以评估组织特异性葡萄糖摄取，以及给予[3-(3)H]葡萄糖以评估胰岛素抑制内源性葡萄糖生成速率（endoRa，肝葡萄糖生成的标志物）和刺激全身葡萄糖消失速率（Rd）的能力。用于代谢疾病基因小鼠模型的胰岛素钳夹技术的小型化推动了糖尿病研究的重大进展。不同实验室进行胰岛素钳夹的方法各不相同。需要注意的是，进行胰岛素钳夹的方式会显著影响所获得的结果。我们已经发表了对清醒小鼠进行胰岛素钳夹的不同方法的全面评估(1)，以及使用各种钳夹技术对四种常用近交系小鼠品系的代谢反应的评估(2)。在此，我们介绍由范德比尔特小鼠代谢表型分析中心（MMPC；网址：www.mc.vanderbilt.edu/mmpc）开发的对清醒、不受约束的小鼠进行胰岛素钳夹的方案。这包括对胰岛素钳夹实验中使用的导管植入方法的描述。范德比尔特MMPC采用的方案利用了独特的双导管系统(3)。一根导管插入颈静脉用于输注。另一根导管插入颈动脉，这样无需限制或处理小鼠即可进行采血。与胰岛素钳夹实验中最常用的从断尾尖采血的方法相比，该技术具有显著优势。与后一种方法不同，从动脉导管采血对小鼠没有压力(1)。我们还描述了使用同位素示踪剂输注评估组织特异性胰岛素作用的方法。我们还为胰岛素钳夹实验获得的结果的恰当呈现提供了指导原则。
