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Mar 2012

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Glucose Production Assay in Primary Mouse Hepatocytes
原代小鼠肝细胞中的葡萄糖生成分析   

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Abstract

Hepatic glucose production is a primary determinant of fasting hyperglycemia in type 2 diabetic patients. Glucagon-cAMP-PKA pathway increases, but insulin-PI3 kinase-Akt pathway suppresses glucose production. This assay aims to evaluate the ability of isolated mouse hepatocytes to release newly synthesized glucose mainly from lactate and pyruvate as the substrates (i.e. gluconeogenesis) under basal, cAMP-, or cAMP plus insulin-treated condition.

Materials and Reagents

  1. Primary mouse hepatocytes
  2. Medium199 (Life Technologies, Invitrogen™, catalog number: 11150-059 )
  3. Fetal bovine serum (FBS)
  4. bicinchoninic acid (BCA)
  5. Penicillin-Streptomycin, Liquid (Life Technologies, Invitrogen™, catalog number: 15140-122 )
  6. PBS+/+ (Sigma-Aldrich, catalog number: D8662 )
  7. Dulbecco’s modified eagle’s medium (DMEM), without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate, powder (Sigma-Aldrich, catalog number: D5030 )
  8. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
  9. Sodium L-lactate (Sigma-Aldrich, catalog number: L7022 )
  10. Sodium pyruvate (Life Technologies, Invitrogen™, catalog number: 11360-070 )
  11. 100 mM MEM sodium pyruvate solution (100x), liquid (Life Technologies, Invitrogen™, catalog number: 11360-070)
  12. 200 mM L-Glutamine (100x), liquid (Life Technologies, Invitrogen™, catalog number: 25030-081 )
  13. 1 M HEPES buffer solution (Life Technologies, Invitrogen™, catalog number: 15630-080 )
  14. pCPT-cAMP (Sigma-Aldrich, catalog number: C3912 )
  15. Insulin (Sigma-Aldrich, catalog number: I9278 )
  16. Autokit glucose (Wako, catalog number: 439-90901 )
  17. BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23227 )
  18. HEPES
  19. Glucose production buffer (see Recipes)

Equipment

  1. iMark Microplate Absorbance Reader (Bio-Rad, catalog number: 168-1135 )
  2. BD BioCoatCollagen I 6-well Plates (BD Biosciences, catalog number: 356400 )

Procedure

  1. Primary mouse hepatocytes were cultured in BD BioCoatCollagen I 6-well plates (1 x 106 cells per well) in Medium199 supplemented with 5% FBS, penicillin (100 units/ml) and streptomycin (100 μg/ml).
  2. 6-48 h after plating, serum-starved overnight in 2 ml/well of Medium199 supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml) without FBS.
  3. Wash the cells by 2 ml/well of warm (37 °C) PBS+/+ twice.
  4. Replace PBS+/+ with 1 ml of glucose production buffer consisting of glucose-free DMEM (pH 7.4) without phenol red supplemented with 20 mM sodium lactate, 2 mM sodium pyruvate, 2 mM L-glutaminie and 15 mM HEPES.
  5. Incubate cells at 37 °C for 6 h with or without 0.1 mM pCPT-cAMP and/or 100 mM insulin, 0.2 ml of medium was collected and the glucose concentration was measured with a colorimetric glucose assay kit.
  6. Collect 0.2 ml of medium from each well.
  7. Measure the glucose concentration with a colorimetric glucose assay kit following manufacturer’s instruction.
  8. Normalized the readings to the total protein content determined from the whole-cell lysates by bicinchoninic acid (BCA) protein assay kit following manufacturer’s instruction.

Recipes

  1. Glucose production buffer (Please note some component come with the DMEM as noted in paresis)
    Components (g/L)
    L-Arginine.HCl 0.084 (contained in DMEM)
    L-Cystine.2HCl 0.0626 (contained in DMEM)
    Glycine 0.030 (contained in DMEM)
    L-Histidine.HCl.H2O 0.042 (contained in DMEM)
    L-Isoleucine 0.105 (contained in DMEM)
    L-Leucine 0.105 (contained in DMEM)
    L-Lysine.HCl 0.146 (contained in DMEM)
    L-Methionine 0.03 (contained in DMEM)
    L-Phenylalanine 0.066 (contained in DMEM)
    L-Serine 0.042 (contained in DMEM)
    L-Threonine 0.095 (contained in DMEM)
    L-Tryptophan 0.016 (contained in DMEM)
    L-Tyrosine.2Na.2H2O 0.10379 (contained in DMEM)
    L-Valine 0.094 (contained in DMEM)
    Choline chloride 0.004 (contained in DMEM)
    Folic acid 0.004 (contained in DMEM)
    Myo-Inositol 0.0072 (contained in DMEM)
    Niacinamide 0.004 (contained in DMEM)
    D-Pantothenic acid (Hemicalcium) 0.004 (contained in DMEM)
    Pyridoxal.HCl 0.004 (contained in DMEM)
    Riboflavin 0.0004 (contained in DMEM)
    Thiamine.HCl 0.004 (contained in DMEM)
    Calcium chloride (anhydrous) 0.2 (contained in DMEM)
    Ferric nitrate.9H2O 0.0001 (contained in DMEM)
    Magnesium sulfate (Anhydrous) 0.09767 (contained in DMEM)
    Potassium chloride 0.4 (contained in DMEM)
    Sodium chloride 6.4 (contained in DMEM)
    Sodium phosphate monobasic (Anhydrous) 0.109 (contained in DMEM)
    L-Glutamine 0.584
    Sodium bicarbonate 3.7
    Sodium lactate 2.24
    Sodium pyruvate 0.22
    HEPES 3.575
    Adjust pH to 7.3, filter using a 0.45 μm filter and store at 4 °C.

Acknowledgments

This protocol was adapted from Sakai et al. (2012). The development of this protocol was supported by a Grant-in-Aid for Creative Scientific Research (to M.K.) and a Grant-in-Aid for Scientific Research (C) (21591155 to M.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a grant from the National Center for Global Health and Medicine (21S116 to M.M.), a grant from Takeda Science Foundation (to M.M.) and a Novo Nordisk Pharma Insulin Award (to M.M.).

References

  1. Matsumoto, M., Pocai, A., Rossetti, L., Depinho, R. A. and Accili, D. (2007). Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell Metab 6(3): 208-216.
  2. Sakai, M., Matsumoto, M., Tujimura, T., Yongheng, C., Noguchi, T., Inagaki, K., Inoue, H., Hosooka, T., Takazawa, K., Kido, Y., Yasuda, K., Hiramatsu, R., Matsuki, Y. and Kasuga, M. (2012). CITED2 links hormonal signaling to PGC-1alpha acetylation in the regulation of gluconeogenesis. Nat Med 18(4): 612-617.
  3. Yoon, J. C., Puigserver, P., Chen, G., Donovan, J., Wu, Z., Rhee, J., Adelmant, G., Stafford, J., Kahn, C. R., Granner, D. K., Newgard, C. B. and Spiegelman, B. M. (2001). Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413(6852): 131-138.

简介

该实验方案的中文版正在准备中...

材料和试剂

  1. 原代小鼠肝细胞
  2. Medium199(Life Technologies,Invitrogen TM,目录号:11150-059)
  3. 胎牛血清(FBS)
  4. 二辛可宁酸(BCA)
  5. 青霉素 - 链霉素,Liquid(Life Technologies,Invitrogen TM,目录号:15140-122)
  6. PBS (Sigma-Aldrich,目录号:D8662)
  7. Dulbecco's改良的Eagle培养基(DMEM),不含葡萄糖,L-谷氨酰胺,酚红,丙酮酸钠和碳酸氢钠,粉末(Sigma-Aldrich,目录号:D5030)
  8. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  9. L-乳酸钠(Sigma-Aldrich,目录号:L7022)
  10. 丙酮酸钠(Life Technologies,Invitrogen TM,目录号:11360-070)
  11. 100mM MEM丙酮酸钠溶液(100x),液体(Life Technologies,Invitrogen TM,目录号:11360-070)
  12. 200mM L-谷氨酰胺(100x),液体(Life Technologies,Invitrogen TM,目录号:25030-081)
  13. 1 H HEPES缓冲溶液(Life Technologies,Invitrogen TM,目录号:15630-080)
  14. pCPT-cAMP(Sigma-Aldrich,目录号:C3912)
  15. 胰岛素(Sigma-Aldrich,目录号:I9278)
  16. Autokit葡萄糖(Wako,目录号:439-90901)
  17. BCA蛋白测定试剂盒(Thermo Fisher Scientific,目录号:23227)
  18. HEPES
  19. 葡萄糖生产缓冲液(参见配方)

设备

  1. iMark微孔板吸光度读数仪(Bio-Rad,目录号:168-1135)
  2. BD BioCoat 胶原I 6孔板(BD Biosciences,目录号:356400)

程序

  1. 将原代小鼠肝细胞在补充有5%FBS,青霉素(100单位)的培养基199中在BD BioCoat胶原I 6孔板(每孔1×10 6个细胞)/ml)和链霉素(100μg/ml)
  2. 接种后6-48小时,在补充有青霉素(100单位/ml)和不含FBS的链霉素(100μg/ml)的Medium199的2ml /孔中血清饥饿过夜。
  3. 用2ml /孔的温热(37℃)PBS(+/+)清洗细胞两次。
  4. 用1ml由无酚红的补充有20mM乳酸钠,2mM丙酮酸钠,2mM L-谷氨酰胺和2mM L-谷氨酰胺的不含葡萄糖的DMEM(pH7.4)组成的葡萄糖生产缓冲液替换PBS +/+ 15 mM HEPES。
  5. 在37℃下用或不用0.1mM pCPT-cAMP和/或100mM胰岛素培养细胞6小时,收集0.2ml培养基,并用比色葡萄糖测定试剂盒测量葡萄糖浓度。
  6. 从每个孔收集0.2ml培养基
  7. 使用比色葡萄糖测定试剂盒根据制造商的说明测量葡萄糖浓度
  8. 通过二辛可宁酸(BCA)蛋白测定试剂盒根据制造商的说明书将读数归一化为由全细胞裂解物测定的总蛋白含量。

食谱

  1. 葡萄糖生产缓冲液(请注意,一些组件随附于DMEM中,如paresis所述)
    成分(g/L)
    L-精氨酸 HCl 0.084(包含在DMEM中) L-半胱氨酸 2HCl 0.0626(包含在DMEM中) 甘氨酸0.030(包含在DMEM中)
    L-组氨酸 HCl H O 0.042(包含在DMEM中)
    L-异亮氨酸0.105(包含在DMEM中)
    L-亮氨酸0.105(包含在DMEM中) L-赖氨酸 HCl 0.146(包含在DMEM中) L-甲硫氨酸0.03(包含在DMEM中)
    L-苯丙氨酸0.066(包含在DMEM中)
    L-丝氨酸0.042(包含在DMEM中)
    L-苏氨酸0.095(包含在DMEM中)
    L-色氨酸0.016(包含在DMEM中)
    L - 酪氨酸 2Na 2H O 0.10379(包含在DMEM中)
    L-缬氨酸0.094(包含在DMEM中)
    氯化胆碱0.004(包含在DMEM中)
    叶酸0.004(包含在DMEM中)
    肌醇0.0072(包含在DMEM中)
    烟酰胺0.004(包含在DMEM中)
    D-泛酸(Hemicalcium)0.004(包含在DMEM中) 吡哆醛0.0014(包含在DMEM中) 核黄素0.0004(包含在DMEM中)
    硫胺盐.0.004(包含在DMEM中) 氯化钙(无水)0.2(包含在DMEM中)
    硝酸铁 9H O 0.0001(包含在DMEM中)
    硫酸镁(无水)0.09767(包含在DMEM中)
    氯化钾0.4(包含在DMEM中)
    氯化钠6.4(包含在DMEM中)
    磷酸二氢钠(无水)0.109(包含在DMEM中)
    L-谷氨酰胺0.584
    碳酸氢钠3.7
    乳酸钠2.24
    丙酮酸钠0.22
    HEPES 3.575
    将pH调节至7.3,使用0.45μm过滤器过滤并在4℃下储存

致谢

该方案改编自Sakai等人(2012)。 该协议的开发由创新科学研究助理(授予M.K.)和科学研究助学金(C)(21591155至 MM),日本教育,文化,体育,科学和技术部,国家全球卫生和医学中心(21S116到MM)的资助,武田科学基金会(MM)和Novo Nordisk制药胰岛素奖(MM)。

参考文献

  1. Matsumoto,M.,Pocai,A.,Rossetti,L.,Depinho,R.A。和Accili,D。(2007)。 缺乏肝脏中叉头转录因子Foxo1的小鼠肝脏葡萄糖生成受损的调节。 Cell Metab 6(3):208-216。
  2. Sakai,M.,Matsumoto,M.,Tujimura,T.,Yongheng,C.,Noguchi,T.,Inagaki,K.,Inoue,H.,Hosooka,T.,Takazawa,K.,Kido, Yasuda,K.,Hiramatsu,R.,Matsuki,Y.and Kasuga,M。(2012)。 CITED2将激素信号传递至PGC-1alpha乙酰化,调节糖异生作用。 Nat Med 18(4):612-617。
  3. Yoon,J.C.,Puigserver,P.,Chen,G.,Donovan,J.,Wu,Z.,Rhee, Adelmant,G.,Stafford,J.,Kahn,C.R.,Granner,D.K.,Newgard,C.B。 和Spiegelman,B.M。(2001)。 通过转录共激活因子PGC-1控制肝脏糖异生。 413(6852):131-138。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Matsumoto, M. and Sakai, M. (2012). Glucose Production Assay in Primary Mouse Hepatocytes. Bio-protocol 2(21): e284. DOI: 10.21769/BioProtoc.284.
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Viviane Augusto
Australian National University
Hi, Just a little question related to this protocol... Do you think this assay is sensitive enough to measure glucose production in HepG2 cells plated in a 96 well plate? The reagent I need to expose my cells during incubation period is quite precious and it will be very helpful to me to perform this assay in a small scale. Best regards, Viviane
2018/3/8 20:55:59 回复
Joonbae Seo
CCHMC
I performed glucose production assay.
The glucose concentration is 0.5-2 mg/dL.
Is the concentration normal range or too low?
How can I improve the assay?
Thank you.
2016/7/12 6:24:03 回复
Is glutamine an essential component in the glucose production buffer? And how critical is the pH 7.4 in the media? Is there any impact on glucose production if the pH is 8.0?
2013/2/2 19:48:08 回复
Michihiro Matsumoto
Department of Molecular Metabolic Regulation, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Japan

We think that glutamine is not essential, but we never perform the experiment to prove it. I don't know the impact of pH 8.0 on glucose production.

2013/2/6 1:41:51 回复