AMPK Methods and Protocols

12. Wash carefully the oxygraph chamber after each measure for
    three times with mQ water, then three times with 70% ethanol
    and again three times with mQ water, in order to remove any
    remaining traces of inhibitors.

3.3 Determination
of Oxygen
Consumption Rates
in Isolated
Mitochondria

3.3.1 Isolation
of Mitochondria
from the Liver

The isolation of liver mitochondria is carried out in a cold room at

4 C according to the standard differential centrifugation method

described by Hogeboom [8] and later optimized by Klingenberg

and Slenczka [9].

1. Harvest liver after decapitation of animals (seeNote 12).


2. Transfer in a glass beaker containing ~10 ml of ice-cold
   homogenization buffer, and quickly cut into small pieces with
   scissors.


3. Rinse twice with the same buffer in order to remove red blood
   cells.


4. Transfer in a 30-ml Potter-Elvehjem and start homogenization
   (~15 ups and downs on ice at 500 rpm).


5. Pour the homogenate into a 50-ml plastic tube and spin down
   at 800gfor 10 min (4C).


6. Transfer the supernatant containing mitochondria into another
   50-ml tube, and spin down at 8000gfor 10 min (4C).


7. Discard the supernatant and resuspend carefully the pellet con-
   taining mitochondria with the homogenization buffer (first
   add ~5 ml and then complete to 50 ml).


8. Spin down again at 8000gfor 10 min (4C).


9. Discard the supernatant and carefully resuspend the pellet con-
   taining mitochondria in 500μl of homogenization buffer sup-
   plemented with 1% fatty acid-free BSA. The final mitochondria
   suspension is kept in an Eppendorf tube on ice until use (within
   a maximal window of ~8 h;seeNote 13).

The determination of mitochondrial protein concentration

(usually ~40 mg/ml for rat liver) can be achieved using the bicinch-

oninic acid assay [10] with BSA as a standard.

3.3.2 Measurement
of Oxygen
Consumption Rate

1. Calibrate the Clark-type polarographic oxygen electrode, as
   described in Subheading3.1.1(seeNote 14).


2. Add 1 mg/ml of isolated mitochondria from whole liver or
   cells into the oxygraph chamber containing 2 ml of the KCl
   medium, as previously reported [11–13].


3. After equilibration for 30 s, the mitochondrial respiratory rate
   is monitored in the presence of various substrates and inhibi-
   tors, as described in Subheading3.2 (steps 5– 12 ) for permea-
   bilized hepatocytes.

Methods for Assessing Mitochondrial OXPHOS 283