Evaluation of insulin sensitivity by hyperinsulinemic-euglycemic clamps using stable isotope-labeled glucose.
Evaluation of insulin sensitivity by hyperinsulinemic-euglycemic clamps using stable isotope-labeled glucose.
复制标题
使用稳定同位素标记的葡萄糖通过高胰岛素正常血糖钳评估胰岛素敏感性
DOI：
10.1038/s41421-018-0016-3
复制
发表时间：
2018
期刊：
影响因子：
33.5
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Zhang Y;Xu L;Liu X;Wang Y
胰岛素抵抗是肥胖、非酒精性脂肪肝（NAFLD）和2型糖尿病（T2 D）等代谢性疾病发病机制的关键因素1。多年来，高胰岛素-正葡萄糖钳夹技术一直被用作精确测量体内胰岛素作用的“金标准”方法2。它被广泛用于人类，狗，大鼠和小鼠。在钳夹期间，常规地用示踪剂3评估葡萄糖动力学，包括小鼠中内源性葡萄糖产生和处置的速率。放射性示踪剂[3- 3 H]葡萄糖是常用的，因为它是敏感的和无质量的，但它是有害的，我们的环境，不能用于人类，因为它是危险的，如果引入体内3。因此，医学研究已转向稳定同位素作为替代示踪剂。虽然许多研究已经成功地建立了在人类中使用稳定同位素的钳夹方法，但由于质谱法的局限性，还没有开发出用于实验室小鼠的方法，这需要注入大剂量的稳定同位素和大量的血液4-8。在这项研究中，我们成功地设计了一种灵敏的方法，使用[6，6- 2 H]葡萄糖作为示踪剂在小鼠中。[6，6- 2 H]葡萄糖是一种稳定的（非放射性）天然存在的同位素，没有已知的有害影响，与正常葡萄糖5的代谢作用相似。使用高分辨率质谱仪可以将其与葡萄糖的天然同位素异构体（即具有其他同位素精细结构）区分开来。为了建立这种方法，我们在高脂饮食（HFD）诱导的肥胖小鼠模型中对其进行了测试，该模型是众所周知的并广泛用于代谢研究9-12。与用常规饮食（RD）喂养的小鼠相比，HFD诱导的肥胖小鼠具有显著更高的体重、血浆胰岛素水平、通过丙酮酸耐量试验（PTT）测量的葡萄糖产生、通过葡萄糖耐量试验（GTT）评估的葡萄糖耐受不良和通过胰岛素耐量试验（ITT）评估的胰岛素不敏感性（图la-e）。所有结果表明，与RD喂养的动物相比，HFD喂养的小鼠的葡萄糖产生和胰岛素抵抗显著增加。为了评价体内胰岛素作用和葡萄糖代谢，我们使用[6，6- 2 H]葡萄糖作为示踪剂进行了高胰岛素-正常血糖钳夹研究（图1f，g）。通过导管推注[6，6- 2 H]葡萄糖（600 μg kg− 1），然后以30 μg kg− 1 min− 1的速率持续输注[6，6- 2 H]葡萄糖90 min，以维持稳态条件。在钳夹期间，我们监测血糖水平、血浆胰岛素水平和葡萄糖输注速率（GIR）（图1 h，i，补充图S1 a）。稳态时，HFD喂养小鼠的GIR为12.17±1.09 mg kg− 1 min− 1，远低于RD喂养小鼠的GIR（22.10±1.23 mg kg− 1 min− 1），表明HFD喂养小鼠的胰岛素敏感性降低（图1 i）。在我们的实验中，使用质谱仪同时测量葡萄糖和[6，6- 2 H]葡萄糖的水平。为了区分[6，6- 2 H]葡萄糖与葡萄糖的天然同位素异构体（M+ 2），我们使用了与超高效液相色谱（UPLC）系统耦合的高分辨率质谱仪（Orbitrap）。最近有报道称，Orbitrap机器的质量分辨率为100，000，因此可以解析同位素精细结构13。我们基于140，000的质量分辨率进行了该测定，这使我们能够区分氧-18（18 O），13 C和2 H同位素© The Author（s）2018
Dear Editor, Insulin resistance is a critical factor in the pathogenesis of metabolic diseases such as obesity, nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) 1. For many years, the hyperinsulinemic-euglycemic clamp has been used as a “gold standard” method to accurately measure insulin action in vivo 2. It is widely used in humans, dogs, rats and mice. During the clamp, glucose kinetics, including the rates of endogenous glucose production and disposal in mice, are conventionally assessed with tracers 3. The radioactive tracer [3-3H] glucose is commonly used because it is sensitive and massless, but it is harmful to our environment, and cannot be used in humans because it is hazardous if introduced into the body 3. Therefore, medical research has turned to stable isotopes as alternative tracers. Although many studies have successfully established the clamp method using stable isotopes in humans, no method has been developed for laboratory mice because of the limitations of mass spectrometry, which requires the infusion of a large dose of stable isotope and a large volume of blood 4–8. In this study, we have successfully devised a sensitive method using [6, 6-2H] glucose as a tracer in mice.[6, 6-2H] glucose is a stable (non-radioactive) naturally occurring isotope with no known harmful effects and similar metabolic effects to normal glucose 5. It can be distinguished from natural isotopomers of glucose (ie, with other isotopic fine structures) using a high resolution mass spectrometer. To establish this method, we tested it in a high-fat diet (HFD)-induced obese mouse model, which is well known and widely used in metabolism research 9–12. Compared to mice fed with a regular diet (RD), HFD-induced obese mice had significantly higher body weight, plasma insulin levels, glucose production measured by pyruvate tolerance test (PTT), glucose intolerance evaluated by glucose tolerance test (GTT) and insulin insensitivity assessed by insulin tolerance test (ITT)(Fig. 1 a–e). All the results indicate that glucose production and insulin resistance were dramatically increased in HFD-fed mice compared to RD-fed animals. To evaluate insulin action and glucose metabolism in vivo, we performed hyperinsulinemic-euglycemic clamp studies using [6, 6-2H] glucose as a tracer (Fig. 1 f, g). A bolus of [6, 6-2H] glucose (600 μg kg− 1) was administered via catheter followed by continuous infusion of [6, 6-2H] glucose at the rate of 30 μg kg− 1 min− 1 for 90 min to maintain steady-state conditions. During the clamp, we monitored the blood glucose levels, plasma insulin levels and glucose infusion rate (GIR)(Fig. 1 h, i, Supplementary Figure S1a). At the steady state, GIR in HFD-fed mice is 12.17±1.09 mg kg− 1 min− 1, which is much lower than that in RD-fed mice (22.10±1.23 mg kg− 1 min− 1), indicating that insulin sensitivity is decreased in HFD-fed mice (Fig. 1 i). In our experiments, levels of glucose and [6, 6-2H] glucose were simultaneously measured using mass spectrometer. To distinguish [6, 6-2H] glucose from natural isotopomers of glucose (M+ 2), we used a high resolution mass spectrometer (Orbitrap) coupled with an ultra-high performance liquid chromatography (UPLC) system. It was recently reported that the Orbitrap machine has a mass resolution of 100,000, and can therefore resolve isotopic fine structures 13. We performed this assay based on a mass resolution of 140,000, which enables us to differentiate oxygen-18 (18O), 13C and 2H isotopes of© The Author (s) 2018
登录
查看更多内容
影响因子：
3.3
作者：
Friedlander, AL;Casazza, GA;Brooks, GA
通讯作者：
Brooks, GA
影响因子：
64.5
作者：
Perry RJ;Camporez JG;Kursawe R;Titchenell PM;Zhang D;Perry CJ;Jurczak MJ;Abudukadier A;Han MS;Zhang XM;Ruan HB;Yang X;Caprio S;Kaech SM;Sul HS;Birnbaum MJ;Davis RJ;Cline GW;Petersen KF;Shulman GI
通讯作者：
Shulman GI
DOI：
10.1097/qai.0b013e3181b03214
发表时间：
2009-10-01
期刊：
Journal of acquired immune deficiency syndromes (1999)
影响因子：
--
作者：
Lee GA;Schwarz JM;Patzek S;Kim S;Dyachenko A;Wen M;Mulligan K;Schambelan M;Grunfeld C
通讯作者：
Grunfeld C
影响因子：
--
作者：
Lutz, Thomas A;Woods, Stephen C
通讯作者：
Woods, Stephen C
影响因子：
64.5
作者：
Samuel VT;Shulman GI
通讯作者：
Shulman GI