AMPK Methods and Protocols

Primary Neuronal Culture Preparation

6. Dissect and isolate mouse cortical and/or hippocampal neu-
   rons using an enzymatic dissociation procedure (seeNote 7).


7. Seed 120,000 cells in 21 wells (seeNote 8and Fig.3), while
   add only medium in the remaining three wells (i.e., position
   A1, C4, D6). The empty wells are required as background
   signal during the assay.


8. Let cells differentiate for 14–17 days in vitro (DIV) before
   running the Seahorse XFe test.

Fig. 3Test for optimal cell density determination. In this test, primary neurons were plated at a density of
50,000, 75,000, or 100,000 cells per well and tested between 14 and 17 DIV. (a) ECAR (expressed in
mpH/min) profile, reflecting the glycolytic flux upon sequential injection of 10 mM glucose, 1μM oligomycin,
and 150 mM 2DG, as indicated byarrows.(b) OCR (expressed in pmoles/min) calculated as mitochondrial
respiration at the basal metabolic state of neurons. Data are meanSD ofn¼4–6. One-way ANOVA, post
hoc test Bonferroni **p<0.05, ***p<0.01

Table 4
Examples of AMPK modulators

Drug Mode of action Ref.

AICAR AIACR is converted by adenosine kinase to AICA ribotide (ZMP). ZMP,

an AMP mimetic, allosterically activating AMPK by binding its

γ-subunit

[20]

A-769662, 991 A-769662 and 991 reversibly binds at an interface between theβ-subunit

carbohydrate-binding module (CBM) and the kinase domain of the

α-subunit. They preferentially act on AMPK containingβ1-subunit

[21, 22]

Compound

2/compound 13

Compound 2 allosterically activates AMPK by binding theγ-subunit. It

activates more efficiently AMPK containingα1-subunit

[23]

MT47-100 MT47-100 activates AMPK containingβ1-subunit, while it inhibits the

AMPK containingβ2-subunit

[24]

Compound C Compound C is an ATP-competitive inhibitor of AMPK [25]

Seahorse Analysis of AMPK-Regulated Metabolic Fluxes 295