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Biology

葡萄糖摄取测量和对胰岛素刺激的反应<em>体外</em>培养的人类初级肌管

Published: June 25, 2017
doi:
10.3791/55743
, , , , , ,
1CarMeN Laboratory, INSERM U1060, INRA 1397,University of Lyon, 2Department of digestive and bariatric surgery, Obesity Integrated Center, University Hospital of Edouard Herriot, Hospices Civils de Lyon,Lyon 1 University, 3Division of Endocrinology, Diabetes, Hypertension and Nutrition, Department of Clinical Medicine, Faculty of Medicine,University of Geneva

Summary

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。

Abstract

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。

Introduction

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>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 。

Protocol

1.细胞培养基和溶液的制备 培养基的制备 通过补充Ham'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）。 通过用谷氨酰胺（2mM），青霉素/链霉素（最终5μg/ mL）和2％FCS补充Dulbecco's Modified Eagle培养基（DMEM）来制备分化培养基（DM）。 葡萄糖摄取溶液的制备…

Representative Results

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?…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

Human primary muscle cell In house preparation from human skeletal muscle biopsies In house preparation from human skeletal muscle biopsies If not available, use commercial source
Human primary muscle cell Promocell C-12530 Should be cultured with associated media C23060 and C23061
6-well plate Corning 356400 BioCoat Collagen I Multiwell Plates
Ham's F10 Dutscher L0145-500 1 g/l glucose
Glutamine Dutscher X0551-100
penicilin/streptomycin 100x Thermo fisher scientific 15140122
Serum substitute UltroserG Pall France 15950.017 serum substitute in text
DMEM low glucose Dutscher L0064-500 1 g/l glucose
Fetal Calf Serum Eurobio CVFSVF00-01
Dulbecco's Phosphate-Buffered Saline Dutscher L0625-500 Contains Mg2+ (0.5 mM) and Ca2+ (0.9 mM)
Insulin solution human Sigma-Aldrich I9278
2-deoxy-D-glucose  Sigma-Aldrich D6134
Albumin bovine euromedex 04-100-812-E
fatty acid-free BSA Roche 10,775,835,001
palmitate Sigma-Aldrich P0500
Deoxy-D-glucose, 2-[1,2-3H (N)] PerkinElmer NET328A001MC Specific Activity: 5-10Ci (185-370GBq)/mmol, 1mCi (37MBq
Cytochalasin B Sigma-Aldrich c2743
PICO PRIAS VIAL 6ml PerkinElmer 6000192
ultima gold MW CA  PerkinElmer 6013159 scintillation liquid
bêta counter  PerkinElmer 2900TR
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References

  1. Stump, C. S., Henriksen, E. J., Wei, Y., Sowers, J. R. The metabolic syndrome: role of skeletal muscle metabolism. Ann Med. 38 (6), 389-402 (2006).
  2. DeFronzo, R. A., Tobin, J. D., Andres, R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol. 237 (3), E214-E223 (1979).
  3. Fossum, E., Hoieggen, A., Moan, A., Nordby, G., Kjeldsen, S. E. Insulin sensitivity relates to other cardiovascular risk factors in young men: validation of some modifications of the hyperinsulinaemic, isoglycaemic glucose clamp technique. Blood Press Suppl. 2, 113-119 (1997).
  4. Heise, T., et al. Euglycaemic glucose clamp: what it can and cannot do, and how to do it. Diabetes Obes Metab. 18 (10), 962-972 (2016).
  5. Sell, H., Jensen, J., Eckel, J. Measurement of insulin sensitivity in skeletal muscle in vitro. Methods Mol Biol. 933, 255-263 (2012).
  6. Sarabia, V., Lam, L., Burdett, E., Leiter, L. A., Klip, A. Glucose transport in human skeletal muscle cells in culture. Stimulation by insulin and metformin. J Clin Invest. 90 (4), 1386-1395 (1992).
  7. Sarabia, V., Ramlal, T., Klip, A. Glucose uptake in human and animal muscle cells in culture. Biochem Cell Biol. 68 (2), 536-542 (1990).
  8. Richter, E. A., Hargreaves, M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev. 93 (3), 993-1017 (2013).
  9. Gaster, M., Kristensen, S. R., Beck-Nielsen, H., Schroder, H. D. A cellular model system of differentiated human myotubes. Apmis. 109 (11), 735-744 (2001).
  10. Bouzakri, K., et al. Reduced activation of phosphatidylinositol-3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes. Diabetes. 52 (6), 1319-1325 (2003).
  11. Scheele, C., et al. Satellite cells derived from obese humans with type 2 diabetes and differentiated into myocytes in vitro exhibit abnormal response to IL-6. PLoS One. 7 (6), e39657 (2012).
  12. Jackson, S., et al. Decreased insulin responsiveness of glucose uptake in cultured human skeletal muscle cells from insulin-resistant nondiabetic relatives of type 2 diabetic families. Diabetes. 49 (7), 1169-1177 (2000).
  13. Aas, V., et al. Are cultured human myotubes far from home?. Cell Tissue Res. 354 (3), 671-682 (2013).
  14. Bakke, S. S., et al. Myotubes from severely obese type 2 diabetic subjects accumulate less lipids and show higher lipolytic rate than myotubes from severely obese non-diabetic subjects. PLoS One. 10 (3), e0119556 (2015).
  15. Stuart, C. A., et al. Hexose transporter mRNAs for GLUT4, GLUT5, and GLUT12 predominate in human muscle. Am J Physiol Endocrinol Metab. 291 (5), E1067-E1073 (2006).
  16. Al-Khalili, L., et al. Insulin action in cultured human skeletal muscle cells during differentiation: assessment of cell surface GLUT4 and GLUT1 content. Cell Mol Life Sci. 60 (5), 991-998 (2003).
  17. Tsuka, S., et al. Promotion of insulin-induced glucose uptake in C2C12 myotubes by osteocalcin. Biochem Biophys Res Commun. 459 (3), 437-442 (2015).
  18. Gorbunov, E. A., Nicoll, J., Myslivets, A. A., Kachaeva, E. V., Tarasov, S. A. Subetta Enhances Sensitivity of Human Muscle Cells to Insulin. Bull Exp Biol Med. 159 (4), 463-465 (2015).
  19. Breen, D. M., Sanli, T., Giacca, A., Tsiani, E. Stimulation of muscle cell glucose uptake by resveratrol through sirtuins and AMPK. Biochem Biophys Res Commun. 374 (1), 117-122 (2008).
  20. Pinnamaneni, S. K., Southgate, R. J., Febbraio, M. A., Watt, M. J. Stearoyl CoA desaturase 1 is elevated in obesity but protects against fatty acid-induced skeletal muscle insulin resistance in vitro. Diabetologia. 49 (12), 3027-3037 (2006).
  21. Gastebois, C., et al. Transition from physical activity to inactivity increases skeletal muscle miR-148b content and triggers insulin resistance. Physiol Rep. 4 (17), (2016).
  22. Naimi, M., Tsakiridis, T., Stamatatos, T. C., Alexandropoulos, D. I., Tsiani, E. Increased skeletal muscle glucose uptake by rosemary extract through AMPK activation. Appl Physiol Nutr Metab. 40 (4), 407-413 (2015).
  23. Feng, Y. Z., et al. PPARdelta activation in human myotubes increases mitochondrial fatty acid oxidative capacity and reduces glucose utilization by a switch in substrate preference. Arch Physiol Biochem. 120 (1), 12-21 (2014).
  24. Perrin, L., et al. Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion. Mol Metab. 4 (11), 834-845 (2015).
  25. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248-254 (1976).
  26. Bouzakri, K., et al. Malonyl CoenzymeA decarboxylase regulates lipid and glucose metabolism in human skeletal muscle. Diabetes. 57 (6), 1508-1516 (2008).
  27. Shemyakin, A., et al. Endothelin-1 reduces glucose uptake in human skeletal muscle in vivo and in vitro. Diabetes. 60 (8), 2061-2067 (2011).
  28. Alkhateeb, H., Chabowski, A., Glatz, J. F., Luiken, J. F., Bonen, A. Two phases of palmitate-induced insulin resistance in skeletal muscle: impaired GLUT4 translocation is followed by a reduced GLUT4 intrinsic activity. Am J Physiol Endocrinol Metab. 293 (3), E783-E793 (2007).
  29. Coll, T., et al. Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. J Biol Chem. 283 (17), 11107-11116 (2008).
  30. Gaster, M., Rustan, A. C., Beck-Nielsen, H. Differential utilization of saturated palmitate and unsaturated oleate: evidence from cultured myotubes. Diabetes. 54 (3), 648-656 (2005).
  31. Hage Hassan, R., et al. Endoplasmic reticulum stress does not mediate palmitate-induced insulin resistance in mouse and human muscle cells. Diabetologia. 55 (1), 204-214 (2012).
  32. Haghani, K., Pashaei, S., Vakili, S., Taheripak, G., Bakhtiyari, S. TNF-alpha knockdown alleviates palmitate-induced insulin resistance in C2C12 skeletal muscle cells. Biochem Biophys Res Commun. 460 (4), 977-982 (2015).
  33. Hommelberg, P. P., et al. Palmitate-induced skeletal muscle insulin resistance does not require NF-kappaB activation. Cell Mol Life Sci. 68 (7), 1215-1225 (2011).
  34. Yang, M., et al. Saturated fatty acid palmitate-induced insulin resistance is accompanied with myotube loss and the impaired expression of health benefit myokine genes in C2C12 myotubes. Lipids Health Dis. 12, 104 (2013).
  35. Peng, G., et al. Oleate blocks palmitate-induced abnormal lipid distribution, endoplasmic reticulum expansion and stress, and insulin resistance in skeletal muscle. Endocrinology. 152 (6), 2206-2218 (2011).
  36. Lambernd, S., et al. Contractile activity of human skeletal muscle cells prevents insulin resistance by inhibiting pro-inflammatory signalling pathways. Diabetologia. 55 (4), 1128-1139 (2012).
  37. Nikolic, N., et al. Electrical pulse stimulation of cultured human skeletal muscle cells as an in vitro model of exercise. PLoS One. 7 (3), e33203 (2012).
  38. Hsu, F. L., et al. Antidiabetic effects of pterosin A, a small-molecular-weight natural product, on diabetic mouse models. Diabetes. 62 (2), 628-638 (2013).
  39. Zou, C., Wang, Y., Shen, Z. 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys Methods. 64 (3), 207-215 (2005).
  40. Catalano, K. J., et al. Insulin resistance induced by hyperinsulinemia coincides with a persistent alteration at the insulin receptor tyrosine kinase domain. PLoS One. 9 (9), e108693 (2014).
  41. Liu, H. Y., et al. Insulin is a stronger inducer of insulin resistance than hyperglycemia in mice with type 1 diabetes mellitus (T1DM). J Biol Chem. 284 (40), 27090-27100 (2009).
  42. Renstrom, F., Buren, J., Svensson, M., Eriksson, J. W. Insulin resistance induced by high glucose and high insulin precedes insulin receptor substrate 1 protein depletion in human adipocytes. Metabolism. 56 (2), 190-198 (2007).

Tags

Glucose UptakeInsulin StimulationHuman Primary MyotubesMuscle MetabolismInsulin SensitivityProliferation MediumDifferentiation Medium2DGCytochalasin BDMSOMuscle Satellite CellsCell Culture
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Chanon, S., Durand, C., Vieille-Marchiset, A., Robert, M., Dibner, C., Simon, C., Lefai, E. Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes. J. Vis. Exp. (124), e55743, doi:10.3791/55743 (2017).
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