4 methylumbelliferyl α d glucopyranoside

Gemcitabine was purchased from Eli Lilly Japan (Kobe, Japan), dissolved in distilled water, and stored at −20°C until use. Bafilomycin A1 (B1793), 4‐Methylumbelliferyl α‐D‐glucopyranoside (M9766) were purchased from Sigma–Aldrich.

GAA activity was measured as previously described.⁸¹ (link) Briefly, 10 μL of sample, either tissue homogenate or plasma, was incubated for 1 h at 37°C with 20 μL of 4-methylumbelliferyl α-d-glucopyranoside (3 mM), diluted in acetate buffer solution (pH 4.65) (Sigma-Aldrich). The reaction was stopped using the carbonate solution, and the fluorescence (λ_ex 360 nm/λ_em 449 nm) was read with an EnSpire alpha plate reader (PerkinElmer, Waltham, MA). A standard curve was prepared using 4-methylumbelliferone, diluted in 0.5 M of carbonate solution (pH 10.5).
Glycogen content was measured indirectly in tissue homogenates as the glucose released after total digestion with Aspergillus niger amyloglucosidase (Sigma-Aldrich, St. Louis, MO, USA). Samples were incubated for 5 min at 95°C and then cooled at 4°C; 25 μL of amyloglucosidase diluted 1:50 in 0.1 M potassium acetate (pH 5.5) was then added to each sample. A control reaction without amyloglucosidase was prepared for each sample. Both sample and control reactions were incubated at 37°C for 90 min. The reaction was stopped by incubating samples for 5 min at 95°C. The glucose released was determined using a glucose assay kit (Sigma-Aldrich, St. Louis, MO, USA) and by measuring resulting absorbance at the EnSpire alpha plate reader (PerkinElmer, Waltham, MA, USA) at 540 nm.

The α-glucosidase
inhibitory activity assay was based on a standard fluorometric assay
as previously described^{63 (link)} using human lysosomal
α-glucosidase-overexpressing preparations from transiently transfected
COS-7 cells.^{64 (link)} Briefly, the inhibitor was
premixed with the appropriate amount of α-glucosidase-overexpressing
homogenates (10–30 μg of protein) and incubated for 10
min at 37 °C. Except in the cases of αGA1, AGT2, AGT9, and AGT11, which were
dissolved in water, the reactions contained 3.5% dimethyl sulfoxide
from the inhibitor solutions. Subsequently, 4-methyl-umbelliferyl
α-d-glucopyranoside (Sigma-Aldrich, St. Louis, MO)
(final concentration = 1.26 mM) in 0.1 M citrate-phosphate buffer,
pH 4.0, was added in an incubation mixture of 70 μL. The reaction
was incubated for 60 min at 37 °C and was terminated by adding
200 μL of 0.5 M sodium carbonate, pH 10.7, with 0.25 g/L Triton
X-100. The release of the product 4-methylumbelliferone was measured
at 365 nm excitation/450 nm emission. The control samples were prepared
without the inhibitor. The percent inhibition of enzyme activity was
calculated using the following formula where control activity
is the enzyme activity
under the same conditions but without the inhibitor.
The IC₅₀ value is defined as the concentration of compound inhibiting
50% of α-glucosidase activity under the stated assay conditions.