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Analysis of Mouse Liver Glycogen Content
小鼠肝糖原含量检测   

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Abstract

Liver is the major site for glycogen storage. Glycogen content can be significantly altered upon disruption of glucose homeostasis in metabolic syndromes, such as diabetes. Glycogen content can be determined by an acid-hydrolysis method (Passonneau and Lauderdale, 1974). Basically, glucose, the hydrolysis product of glycogen, is converted into glucose-6-phosphate (G-6-P) by hexokinase in the presence of ATP. With the supply of NADP, G-6-P is further converted into 6-phosphogluconic acid by G-6-P dehydrogenase (G-6-PDH), while production of NADPH can be measured spectrophotometrically. Our lab has used this method to demonstrate that liver glycogen levels are significantly elevated in diabetic Perk knockout mice (Zhang et al., 2002).

Materials and Reagents

  1. Hydrochloric acid (Sigma-Aldrich, catalog number: 653799 )
  2. Sodium hydroxide
  3. Glucose (HK) assay reagent (Sigma-Aldrich, catalog number: G2020 )
  4. Beta-D(+)-glucose (Sigma-Aldrich, catalog number: G5767 )
  5. ddH2O
  6. Liquid nitrogen
  7. 2.0 M HCl
  8. 2.0 M NaOH

Equipment

  1. Centrifuges (Eppendorf, Model 5415D )
  2. Cryogenic vials (Thermo Fisher Scientific, catalog number: 5000-0012 )
  3. 2 ml Eppendorf tube with locking lid (Thermo Fisher Scientific, catalog number: 02-681-299 )
  4. Spectra Max 384 spectrophotometer (Molecular Devices)
  5. Balance
  6. Quartz cuvette
  7. Scissors

Procedure

  1. Liver sample preparation
    1. Sacrifice non-fasted animals and isolate liver slices. Transfer samples into cryogenic vials, flash-frozen in liquid nitrogen, and stored at -80 °C before use.
      Notes:
      1. Collect liver samples in the afternoon or during night when animals are well fed.
      2. Try to collect samples from around the same region (I typically used the right liver lobe).
      3. Record the growth and nutrition status of study subjects (body weight, water consumption, etc).
      4. Use the frozen samples within a month (I have never addressed whether degradation of glycogen could occur during long-term storage).
    2. For each single sample, prepare 0.5 ml of 2.0 M HCl in a 2 ml Eppendorf tube with locking lid. As the control to measure free glucose, substitute 2.0 M HCl with 2.0 M NaOH. Prepare and label enough tubes with either HCl or NaOH.
    3. Heat the tubes in boiling water for ~ 3 to 5 min.
    4. Centrifuge briefly, and then wipe the tubes and measure the weights on an analytical balance.
    5. Prepare frozen liver samples on dry ice. Transfer ~10 mg to 20 mg samples into the above tubes with hot HCl or NaOH. Measure the weights again on balance.
    6. Seal the tubes tightly and boil the samples in water for 1 h. To achieve complete hydrolysis, mince the liver samples within ~5 min with a scissors, and then shake the tubes vigorously every 10 min during the whole process.
    7. Cool samples on bench to room temperature (RT) and centrifuge briefly. Reconstitute original weights by adding ddH2O.
    8. Neutralize the hydrolysis products with 0.5 ml of 2.0 M NaOH or HCl, accordingly.
    9. Vortex samples vigorously and then centrifuge at maximum speed (i.e., ~ 22,000 x g) for 10 min.

  2. Glucose level determination - Glucose concentration of the hydrolysis product can be determined using the Glucose (HK) assay reagent.
    1. Reconstitute assay reagent with 20 ml ddH2O, and use this reagent within two months.
    2. For each single sample, prepare 1 ml assay reagent in an Eppendorf tube, and then add 10 μl supernatant of hydrolysis product. Meanwhile, use 10 μl freshly prepared 0.5 mM beta-D(+)-glucose as the standard, and 10 μl ddH2O as the blank.
    3. Incubated at RT for 5 min.
    4. Measure absorbance at 340 nm on a Spectra Max 384 spectrophotometer, using a Quartz cuvette.

  3. Liver glycogen content is determined as:
    Abs (sample)/Abs (standard) x Concentration (standard) (i.e., 0.5 mM) x Volume (standard) (i.e., 0.01 ml) x Total volume (i.e., 1.0 ml)/Volume (sample) (i.e., 0.01 ml)/ Weight (sample) (mg) x 1000
    Unit = micromoles glucosyl units/ per gram wet liver weight.

Notes

Please note that gender, age, genetic background, and particularly, feeding status, which can be affected by housing light-dark cycle, can affect glycogen levels in the liver. Animals fed ad libitum should be used for this type of experiment.

Recipes

  1. 2.0 M HCl
  2. 2.0 M NaOH
  3. Glucose (HK) assay reagent

Acknowledgments

This protocol was adapted from the previously described work of Passonneau and Lauderdale (Passonneau and Lauderdale, 1974). PZ was supported by a research assistantship in the Cavener lab at the Pennsylvania State University. This work was supported by an NIH R01 grant awarded to DC.

References

  1. Passonneau, J. V. and Lauderdale, V. R. (1974). A comparison of three methods of glycogen measurement in tissues. Anal Biochem 60(2): 405-412.
  2. Zhang, P., McGrath, B., Li, S., Frank, A., Zambito, F., Reinert, J., Gannon, M., Ma, K., McNaughton, K. and Cavener, D. R. (2002). The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas. Mol Cell Biol 22(11): 3864-3874.

简介

肝是糖原储存的主要部位。 糖原含量可以在代谢综合征例如糖尿病中的葡萄糖体内平衡破坏时显着改变。 糖原含量可以通过酸水解方法测定(Passonneau和Lauderdale,1974)。 基本上,糖蛋白的水解产物葡萄糖在ATP的存在下通过己糖激酶转化为葡萄糖-6-磷酸(G-6-P)。 通过供应NADP,G-6-P通过G-6-P脱氢酶(G-6-PDH)进一步转化为6-磷酸葡萄糖酸,同时可以通过分光光度法测量NADPH的产生。 我们的实验室已经使用这种方法证明在糖尿病Perk敲除小鼠中肝糖原水平显着升高(Zhang等人,2002)。

材料和试剂

  1. 盐酸(Sigma-Aldrich,目录号:653799)
  2. 氢氧化钠
  3. 葡萄糖(HK)测定试剂(Sigma-Aldrich,目录号:G2020)
  4. β-D(+) - 葡萄糖(Sigma-Aldrich,目录号:G5767)
  5. ddH sub 2 O
  6. 液氮
  7. 2.0 M HCl
  8. 2.0 M NaOH

设备

  1. 离心机(Eppendorf,Model 5415D)
  2. 低温小瓶(Thermo Fisher Scientific,目录号:5000-0012)
  3. 2ml具有锁定盖的Eppendorf管(Thermo Fisher Scientific,目录号:02-681-299)
  4. Spectra Max 384分光光度计(Molecular Devices)
  5. 余额
  6. 石英比色皿
  7. 剪刀

程序

  1. 肝样品制备
    1. 牺牲非禁食动物并分离肝切片。 将样品转移到低温小瓶中,在液氮中快速冷冻,并在使用前储存在-80℃ 注意:
      1. 在下午或晚上收集肝脏样本,当动物喂食良好。
      2. 尝试从同一区域收集样品(我通常使用右肝叶)。
      3. 记录研究受试者的生长和营养状况(体重,耗水量等)。
      4. 在一个月内使用冷冻的样品(我从来没有提到在长期储存过程中是否会发生糖原的降解)。
    2. 对于每个单个样品,在带有锁定盖的2ml Eppendorf管中制备0.5ml的2.0M HCl。作为测量游离葡萄糖的对照,用2.0M NaOH代替2.0M HCl。用HCl或NaOH制备和标记足够的管子
    3. 在沸水中加热管子约3至5分钟。
    4. 短暂离心,然后擦拭试管,并在分析天平上测量重量
    5. 在干冰上制备冷冻肝脏样品。用热HCl或NaOH将约10mg至20mg样品转移到上述管中。在天平上再次测量重量。
    6. 紧紧密封管,将样品在水中煮沸1小时。为了实现完全水解,用剪刀在〜5分钟内切碎肝脏样品,然后每10分钟剧烈摇动管 整个过程
    7. 将样品在台上冷却至室温(RT),并短暂离心。 通过添加ddH 2重建原始权重。
    8. 因此,用0.5ml的2.0M NaOH或HCl中和水解产物
    9. 涡旋样品,然后以最大速度离心(,即〜22,000in×g )10分钟。

  2. 葡萄糖水平测定 - 可以使用葡萄糖(HK)测定试剂测定水解产物的葡萄糖浓度。
    1. 用20mL ddH 2 O重建测定试剂,并在两个月内使用该试剂。
    2. 对于每个单个样品,在Eppendorf管中制备1ml测定试剂,然后加入10μl水解产物的上清液。 同时,使用10μl新制备的0.5mMβ-D(+) - 葡萄糖作为标准品,和10μlddH 2 O作为空白。
    3. 在室温下孵育5分钟。
    4. 使用石英比色杯在Spectra Max 384分光光度计上测量340nm处的吸光度

  3. 肝糖原含量测定为:
    Abs(样品)/Abs(标准)x浓度(标准)(即,0.5mM)x体积(标准)(即0.01ml)x总体积(

笔记

请注意,性别,年龄,遗传背景,特别是喂养状况,可能受房屋光 - 黑暗循环影响,可以影响肝脏中的糖原水平。 自由进食的动物应该用于这种类型的实验。

食谱

  1. 2.0 M HCl
  2. 2.0 M NaOH
  3. 葡萄糖(HK)测定试剂

致谢

该协议改编自Passonneau和Lauderdale的先前描述的工作(Passonneau和Lauderdale,1974)。 PZ由宾夕法尼亚州立大学Cavener实验室的研究助理支持。 这项工作得到了授予DC的NIH R01资助。

参考文献

  1. Passonneau,J.V.and Lauderdale,V.R。(1974)。 组织中糖原测量的三种方法的比较。 /em> 60(2):405-412。
  2. Zhang,P.,McGrath,B.,Li,S.,Frank,A.,Zambito,F.,Reinert,J.,Gannon,M.,Ma,K.,McNaughton,K.and Cavener,DR )。 PERK真核起始因子2α激酶是骨骼系统发育,出生后生长所必需的, 和胰腺的功能和存活力。 Mol Cell Biol 22(11):3864-3874。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, P. (2012). Analysis of Mouse Liver Glycogen Content. Bio-protocol 2(10): e186. DOI: 10.21769/BioProtoc.186.
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sonam9a Chawla
DIPAS DRDO
Hello
I have been using your protocol for estimation of glycogen in liver and heart tissue. However, as recommended by you i use 40-50 mg tissue. Also in the step requiring estimation of free glucose i usually do not capture the tissue glucose content. Is the protocol working fine?? I get extremely low readings in the free glucose estimation which usually do not correlate with the results of my free glucose estimation assay in the tissue homogenate. Kindly comment. Looking forward to your reply

Thanks....
Sonam
10/31/2015 6:34:03 AM Reply
Peichuan Zhang
Calico Life Sciences

Hi Sonam,

Could you please elaborate what you mean by "estimation of free glucose"? This protocol is based on acid-hydrolysis of glycogen in storage tissues, including liver and muscles, to produce free glucose that can be detected using the HK assay. The levels of glycogen could be low in fasted mice. However, according to what I could recall, we could always detect pretty good signals by the HK assay. Please make sure that the tissues are frozen in liquid nitrogen upon harvest, and it would be nice to prepare a glucose standard curve, just to to check the quality of the assay buffer.

Best,
Peichuan

11/12/2015 10:13:40 PM


Damilola Tanimowo
University of Ibadan
Hello Sir, really appreciate your post. I will like your assistance, i am to determine glycogen levels in poultry tissue, but standards are not available here and take between 6-8weeks for shipping after orders have been made. Using the PHESUL method is there a way that glucose could be used as a standard (since the glycogen present will be eventually hydrolised to glucose) for colorimetric readings?
9/12/2014 8:58:24 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Damilola,

Actually, we used glucose (10ul, 0.5mM) as the standard for calculation in the assay. The acid hydrolysis step converts glycogen into free glucose, which then can be measured by the HK assay kit. You can find the assay kit from Sigma http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/gahk20bul.pdf

Best,
P

9/22/2014 10:12:42 AM


Damilola Tanimowo
University of Ibadan

Hello Sir, I would like to know is there a colorimetric determination protocol for betaine and dimethylglicine in blood?

1/14/2015 1:26:20 AM


Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Damilola,

I have never done this type of assay. You may refer to other online protocols, for example, chromatography based
http://www.ncbi.nlm.nih.gov/pubmed/12560353

Best,
P

1/18/2015 1:11:51 PM


Yunbing Ma
LSU Health New Orleans
Hi, thanks again for posting the protocol. I have one question.
After boiling the liver sample in the HCl (or NaOH), the weight is measured and water is added back in case of any evaporation. Then equal amount of NaOH or HCl is added to reneutralize the solution.
Do you test the pH to see whether the solution is actually neutral?
6/11/2014 3:30:03 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Yunbing,

It is not necessary to measure the pH, as you will be using a rather small volume of hydrolysis product for the next step of enzyme reaction, which has a buffer for the reaction.

6/11/2014 6:09:25 PM


Yunbing Ma
LSU Health New Orleans

Yeah..I use 1:200 in 0.05M sodium phosphate buffer (pH 7.4), and still have pH issues 90% of the time..I guess your dilution is around 1:100? (10ul supernatant+1ml assay reagent)

6/11/2014 9:06:52 PM


Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

I'd recommend you to prepare fresh 2M NaOH, and use fresh 2M HCl for the 1st step of acid hydrolysis (dilute from the 12.1M stock solution --- please refer to http://www.sigmaaldrich.com/china-mainland/zh/chemistry/stockroom-reagents/learning-center/technical-library/reagent-concentrations.html). Yes - we used 10ul supernatant for the assay (1.0ml assay buffer with the HK enzyme). Should pH be a problem, you may consider to use less supernatant (e.g., 2ul) and increase the volume of assay solution (e.g., 1.5ml).

6/12/2014 9:22:41 AM


Shilpi Islam
Hirosaki University
Dear Sir
Thanks a lot for repeated cooperation and your valuable suggestions. I could not understand that glucosyl unit containing only glycogen or ester of glycogen (glycogen + glucose). I need to express my result as the concentration of glucose mg / g or glycogen mg/g wet tissue for comparison to others results of sheep liver. So,I need the conversation ration of glucosyl unit to glucose or only glycogen content. Please, if possible inform me.

Shilpi Islam
5/26/2014 8:39:24 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Shilpi,

Acid hydrolysis of glycogen will yield glucose monomers, which will be converted further into G-6-P by hexokinase in the kit. The glucosyl unit, as expressed in the equation, refers to a glucose monomer. 1 umole of glucosyl unit is roughly 180ug (D-glucose, MW=180.2).

6/11/2014 6:06:29 PM


Shilpi Islam
Hirosaki University
Dear Sir
Thanks a lot for your valuable information. I followed your protocol as per your recommendation. In normal condition, sheep liver glycogen concentration is below of 100 mg/g wet liver wt. But my glycogen value was 1200-1500 mg/g wet liver wt.which too large. I boiled about 20 mg liver sample in hot 2M HCL for 1 hour and neutralized by 2M NaOH. Please inform me.




5/22/2014 7:00:35 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Shilpi, we typically analyzed liver glycogen levels for mice that had been fasted overnight. I'd recommend you to check the feeding status for your study subjects, which would have strong effects on their liver glycogen contents. Meanwhile, could you please check and make sure that you use the right amount of glucose standard for the calculation?

Best,
Peichuan

5/23/2014 4:33:02 AM


Shilpi Islam
Hirosaki University
Dear sir
I am Shilpi Islam. Thank you for your previous solution. I have another inquire that after hemolysis of liver sample supernatant hydrolyzed solution will be used for the glycogen content. Can I store that supernatant solution for further use? If that is possible, so, what condition (storage temperature and time longevity)is require? If sample weight is more than 20 mg than hydrolysis solution (HCL/NaOH) volume (0.5 ml) will be changed or not? Please inform me.
5/11/2014 10:36:45 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Shilpi,

This is a good question. Typically, I'd conduct the HK glucose assay right away once when I have obtained the glycogen hydrolysis product. 10 minutes of boiling is sufficient to kill the enzymes that could possibly degrade glucose, the molecule of our interest. However, long-term storage (for example, @ -20C or -80C with repeated cycles of freezing/thawing) may increase oxidation of glucose to gluconolactone, and thus, bring more deviations to the final assay. I'd recommend you to store the products (protect them from light) @ 4C if you need to wait to have all the samples ready for the assays.

Best,
Peichuan

5/12/2014 5:12:59 PM


Shilpi Islam
Hirosaki University
Dear Sir
I am a student. I have sheep liver and kidney samples. Can I determine my samples glycogen by this reagents? please inform me.
4/23/2014 11:12:02 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi, we have never used this protocol to analyze samples other than mouse livers. I assume that it would work as well. Glycogen is also present in the kidney in a small amount --- I didn't find out how much it is. You may check this reference as well:

Bulletin of Environmental Contamination and Toxicology
JAN.–APRIL 1979, Volume 21, Issue 1, pp 269-272
Levels of blood glucose and tissue glycogen in two live fish exposed to industrial effluent

A. D. Diwan, H. G. Hingorani, N. Chandrasekhram Naidu

They analyzed fishes, and the relative glycogen abundance in different tissues could provide you with a reference.

BTW, please also be aware that liver glycogen level increases significantly after meals.

Hope this info could be of help to you.

Best,
Peichuan

4/26/2014 8:44:03 AM


Ahmed Abou-Mesalam
Zagazig University
Hello sir
please explain the calculations because i cannot understand it
thanks alot
10/20/2013 12:28:30 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Thank you for the question. The same volume of hydrolysis products and 0.5mM glucose standard solution were used in the final enzymatic assay, and the concentration of glucosyl groups in the hydrolysis product is calculated by normalization of the absorbance of that of the 0.5mM globose. Then the total amount of glucosyl units in the 1.0ml hydrolyzed liver sample is determined, and then used for the calculation of average glucosyl units.

For example, 10mg liver was hydrolyzed and dissolved in 1.0ml solution.
Abs 10ul sample = 0.4
Abs 10ul 0.5mM glucose = 0.8

Then the total amount of glucosyl units in this 1.0ml solution will be:
0.4/0.8 x 0.5 x 1 = 0.25 umol
The average would be 0.25/10 = 0.025 umol glucosyl units/ mg wet liver

I hope this have answered your question.

Best,
Peichuan

10/22/2013 10:56:47 PM


norli arlizan
uitm
i am final year student in uitm and now I'm having problems with the project . I would like to study the level of liver glycogen in the diabetic rats that given a drug metformin and given plant extract.the problem is , to carry out the procedure I have no standard glycogen . Is there is other way todetermine the glycogen level result instead of you have given above?tq
5/8/2013 10:15:34 PM Reply
Good evening Sir/Mam,

I, G.Vinod kumar, working as a research scholar in Department of zoology India. In my resarch work i am going to concentrate on some biochemical aspects i.e., total protein (µg/mg), total glycogen (µg/mg), total carbohydrate (µg/mg), total DNA (µg/mg), total RNA (µg/mg), Total amino acids (µg/g) and GST enzyme quantity mg/unit protein mice tissues.I have some prtocols to quantify the above parameters in mg/ml and (µg/µl). But i would like to take the dry tisisue and estimate the amount of all the above said parameters in units (µg/mg) instead of (mg/ml) or (µg/µl).Hence i am requesting you to help by providing protocols to estimate them in mentioned units. Thanking you.

Sincerely Yours,
mailme.gvk@gmail.com
vinodkumargollapudi@gmail.com
4/15/2012 2:47:53 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi,

I have only used the above protocol to measure glycogen concentrations in the liver. I'm not quite that I fully understand your question though. From the description of your experiments, I believe the only way is to measure each single parameter, using respective methods/kits, and then normalize the quantities to the weight of the dry tissue.

TRIZol (Invitrogen) is supposed to help the isolation of DNA/RNA/protein from just one prep. However, I don't think that the isolate quality is good enough. Otherwise, I'd say that you may consider the most common methods to measure these

Protein - Bradford assay or BCA assay
Carbohydrate - Phenol/sulfuric acid method
DNA/RNA - Tissue extraction kit (Qiagen)
Amino acids - Chromatography
GST - ELISA kit

I don't think that we have all the protocols available here at our portal, and we will try to collect all these protocols.

Best,
Peichuan

4/18/2012 1:24:56 PM