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
Equipment
Procedure
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
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
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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
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.pdfBest,P
Hello Sir, I would like to know is there a colorimetric determination protocol for betaine and dimethylglicine in blood?
Hi Damilola,I have never done this type of assay. You may refer to other online protocols, for example, chromatography basedhttp://www.ncbi.nlm.nih.gov/pubmed/12560353Best,P
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.
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)
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).
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).
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
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
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 ToxicologyJAN.–APRIL 1979, Volume 21, Issue 1, pp 269-272Levels of blood glucose and tissue glycogen in two live fish exposed to industrial effluentA. D. Diwan, H. G. Hingorani, N. Chandrasekhram NaiduThey 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
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 = 0.4Abs 10ul 0.5mM glucose = 0.8Then the total amount of glucosyl units in this 1.0ml solution will be:0.4/0.8 x 0.5 x 1 = 0.25 umolThe average would be 0.25/10 = 0.025 umol glucosyl units/ mg wet liverI hope this have answered your question.Best,Peichuan
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 theseProtein - Bradford assay or BCA assayCarbohydrate - Phenol/sulfuric acid methodDNA/RNA - Tissue extraction kit (Qiagen)Amino acids - ChromatographyGST - ELISA kitI 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