Figure 3. AMPK phosphorylates and stimulates PAK signaling.

(A) Single cell RT-PCR in AgRP neurons

(B–D) Arcuate α2AMPK activity (B) and total and phosphorylated PAK2 (Ser²⁰) (C) and LIMK2 (Thr⁵⁰⁵) (D) in arcuate lysates from fasted and 6-hr refed wildtype mice (n_refed=9 and n_fasted=8).

(E) Immunofluorescence of arcuate p-Thr⁵⁰⁵LIMK2 from fed and 24-hr fasted Npy-hrGFP mice.

(F) Immunoprecipitation of PAK2 and α2AMPK from arcuate lysates of fed wildtype mice.

(G) Phosphorylation of PAK2 (Ser²⁰) and LIMK2 (Thr⁵⁰⁵) in the arcuate of fed wildtype mice following bilateral injection of HA_tag-CA-AMPK adenovirus (n=5).

(H–K) Schematics of cre-independent AAV-DN-AMPK and stereotaxic injection into mediobasal hypothalamus (MBH) (H), immunofluorescence of mCherry (red) and p-Thr⁵⁰⁵LIMK2 (green) from fasted non-viral infected control mice (I) and AAV-DN-AMPK injected mice (J), and the ratio of total and phosphorylated LIMK2 (Thr⁵⁰⁵) in the arcuate lysates as detected with western blot from fasted and 6-hr refed mice following AAV-DN-AMPK injection (K)(n=8).

(L–N) Total and phosphorylated ACC (Ser⁷⁹), PAK2 (Ser²⁰) and LIMK2 (Thr⁵⁰⁵) in GT1-7 cells following glucose starvation or AICAR treatment (L), or transfection of CA-AMPK (n=9) (M), or transfection of PAK2^WT and PAK2^S20A mutants with 1 mM AICAR treatment (N). Proteins are normalized to GAPDH.

Data are mean ± SEM and * indicates p<0.05 with unpaired two-tailed student’s t-test.