glycogen in the skeletal and cardiac muscles of pigs and mice
[16–22]. Hunter et al. [14] demonstrated that the chronic
AMPK activation heightens glucose uptake and increases glucose-
6-phosphate cellular levels, which allosterically activate glycogen
synthase, bypassing the inhibitory effects of its phosphorylation
by AMPK [14]. This has been reported in multiple systems and
organisms. InC. elegans, we have shown that AMPK mutant ani-
mals display decreased levels of glycogen [23, 24], similarly to what
has been previously observed in yeast [25]. However, loss offlcn-1,
the worm homolog of the BHD gene responsible for the Birt-
Hogg-Dube ́neoplastic syndrome in humans, constitutively acti-
vates AMPK and leads to glycogen accumulation [23, 24].
The role of AMPK in the regulation of glycogen metabolism is
a long paradox that needs to be resolved. Glycogen determination
in cells and tissues is therefore an important method that could be
utilized to address this aspect. Here, we describe a quantitative
in vitro glycogen determination biochemical technique using a
colorimetric or fluorimetric method that involves glycogen extrac-
tion from cells and hydrolysis into glucose monomers using amy-
loglucosidase enzyme. Glucose is then oxidized by glucose oxidase
to produceD-gluconic acid and hydrogen peroxide, which interacts
with the kit’s probe leading to a product that could be detected
either in a colorimetric plate format or in fluorimetric plate format.
These kits are widespread and are based on the same strategy. They
are sold at a reasonable price by several commercial companies
worldwide. In Subheading4, we compare this method to other
alternative techniques that could be used for this purpose. We
provide a list of advantages and disadvantages of this method in
comparison to other biochemical titration methods. We also
describe extensively tricks and cautions that should be taken into
consideration while performing this method.2 Materials
2.1 Cell Lysis
and Glycogen
Extraction
- Phosphate buffer solution (PBS): 10 mM phosphate, 137 mM
NaCl, and 2.7 mM KCl. To prepare 1PBS, use 8 g of NaCl,
0.2 g of KCl, 0.2 g of KH 2 PO 4 , 1.15 g of Na 2 HPO 4 , and add
Milli-Q H 2 O up to 1 l. Adjust the pH to 7.4. - 30% potassium hydroxide buffer.
- 95% ethanol.
- Cooled high-speed centrifuge.
- Milli-Q H 2 O.
Biochemical Titration of Glycogen in Cells and Tissues 59