- Handpick islets (seeNote 9) into a 1.5 ml tube, spun for 2 min
at 200g, and resuspend in a small volume (50–100μl per
condition) of mouse islet medium. Equal volumes of the resus-
pended islets are subsequently added into each well. - Incubation is carried out for 48 h.
- Following incubation, lyse islets in TRIzol for RNA extraction
(seeSubheading3.4) or in RIPA buffer for protein analysis (see
Subheading3.3). As hinted above, our studies on the effect of
acute (short-term) modulation of AMPK activity are normally
aimed to assess its impact on insulin secretion. Therefore, we
used the protocol described in Subheading3.3 with minor
modifications as indicated inNote 12.
Modulation of AMPK Using Virus Expression Dominant-
Negative (DN) and Constitutively Active (CA) AMPK Proteins
The group of David Carling designed a constitutively active
AMPK and a dominant-negative inhibitor of endogenous AMPK
[23] which were used by our group [12] to generate adenovirus for
islet infection (seeNote 11). These viruses have been successfully
used to modify AMPK activity in islets applying the following
guidelines:- Handpick 30–100 islets< 100 μm(seeNote 9) per sample
(depending on downstream application) and place in 6-well
plates containing 2.5–3 ml of mouse islet medium. - Use a multiplicity of infection of 50–100 MOI for 16–48 h. An
islet of 100μm diameter will typically contain ~1000 cells.
We have previously demonstrated that changes in AMPK activ-
ity, using both mouse and human islets, can be detected 36 h [12]
or 48 h [24] after infection, respectively.
SeeNotes 13 and 14for additional tips/recommendations.3.3 Measurement
of AMPK Activity
in Isolated Islets
It should be stressed that the demonstration of changes in AMPK
activity requires close attention to several aspects of the islet isola-
tion and incubation protocols that are detailed inNote 9.
SAMS Peptide Assay
AMPK activity is measured using the so-called SAMS peptide
assay, where a synthetic peptide (HMRSAMSGLHLVKRR) is
phosphorylated by AMPK in the presence of radioactiveγATP
[25]. AMPK activity can be assayed from whole cell lysates
(Fig.2a, reproduced from Leclerc et al. 2004 with permission
fromAPS) or after immunoprecipitation using a specific anti-
AMPK subunit antibody [26]. To measure AMPK activity from
whole islet lysates:- Approximately 100–150 islets are required for each sample
(duplicates recommended).
Manipulation and Measurement in Islets 421