- Prepare PBS at required glucose concentration (i.e., 3, 11,
17 mM). - Add 1 ml of PBS in each well of a nontreated 24-well plate.
- Wash islets six times by taking across wells with care.
- Place islets in 1.5 ml microfuge tube, centrifuge at 350gfor
1 min, and remove PBS. - Add 25μl of lysis buffer.
- Aspirate several times to dissociate the islets with a p200 pipette
and incubate on ice for 30 min. - Determine protein concentration using BCA protein kit
(Pierce). - To assay, place in 1.5 ml tube: 10μg of protein extract (seeNote
15 ), 10μlof5assay buffer, and 10μlof5ATP (hot/cold).
Adjust with H 2 O to a total volume of 50μl.
Fig. 2Measurement of AMPK activity in isolated islets. (a) AMPK activity is
modulated by overnight incubation at different glucose concentrations and/or
with AICAR in human islets, as determined by SAMS peptide assay. Reproduced
from Leclerc et al. 2004 with permission fromAPS[24]. (b) In MIN6 cells, AMPK
activity is regulated by glucose and increased by treatment with metformin during
16 h. Increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation were
measured by immunoblotting in MIN6 cells after treatment with metformin
(1 mM). Reproduced from Leclerc et al. 2004 with permission fromAPS[24]422 Aida Martinez-Sanchez et al.