same volume of liquid added. All wells, including the control
wells and any blanks, must have vehicle or compound loaded in
the appropriate ports.- Prepare assay protocol (seeNote 18). Load the sensor cartridge
plate (containing the compounds of interest) and perform
instrument calibration. Replace the utility plate with the cell
culture plate when instructed. Run the assay. - Once the assay is completed, collect the cells. If required,
harvest cells and determine the actual number per well (e.g.,
by counting with a hemocytometer). This number can be used
to normalize the OCR for exact cell number (seeNote 19). - Changes in OCR are indicative of a disruption in mitochon-
drial function and suggest that the compound of interest is
likely to activate AMPK via increases in the AMP/ATP or
ADP/ATP ratios. If an AMPK activator does not change
OCR, it is likely to be acting via a different mechanism. We
have previously characterized a number of AMPK activators in
this way [3]. - The extracellular acidification rate (ECAR), measured simulta-
neously to the OCR, is often used as a measure of the rate of
glycolysis and may be useful for assessing the effects of AMPK
activators that act by inhibiting glycolysis. However, we have
no first-hand experience with that approach.
3.8 Suggested
Methods for Analyzing
AMPK Activation
in Cell Lysates
Once cell lysates have been prepared, some thought should be
given to the most appropriate method of determining the degree
of AMPK activation. There are two widely used approaches:- AMPK immunoprecipitate (IP)-kinase assay (seeSubheading
3.5 in Chapter 5). This radiometric assay measures AMPK
activity due to changes in the covalent modification of
Thr172, the key residue on theαsubunit phosphorylated by
the upstream kinases LKB1 and CaMKK2, and represents a
quantitative, sensitive, and reproducible assay for AMPK acti-
vation. However, it cannot address effects of allosteric activa-
tion, since any allosteric effects due to non-covalent binding of
ligands are lost during the preparation of the
immunoprecipitate. - Western blotting using phosphospecific antibodies that recog-
nize either the phosphorylated, active form of AMPK itself
(pT172) or a phosphorylated downstream target such as
acetyl-CoA carboxylase (pACC; see Note 20) or Raptor
(p-Raptor, phosphorylated on S792). Similar to the IP-kinase
assay, pT172 blots only reflect the degree of AMPK activation
that is due to changes in phosphorylation of Thr172 by the
upstream kinases and ignore any concomitant allosteric activa-
tion. In contrast, pACC and p-Raptor blots should reflect both
Intact Cell Assays to Determine Contributions of AMP-Binding and ADaM Sites 249