P ampk

Total AMPK and p-AMPK protein levels were measured by Western-blot (8 or 12% polyacrylamide gels) in AMN/C-ALD fibroblasts and in Abcd1 KO glial cells, as described (Supplemental Material), using the following antibodies: AMPKα1/2 (T-AMPK, Cell Signaling, #2532) and p-AMPK (Cell Signaling, #2535).

For histopathological examination, paraffin sections (4 μM) from dissected liver and adipose tissues were stained with hematoxylin and eosin. Immunohistochemical staining of PPARγ (Novus, Littleton, CO, USA) and p-AMPK (Cell signaling Tech., MA, USA) was performed using the indirect avidin/biotin-enhanced horseradish peroxidase method. Antigen retrieval was performed after dewaxing and dehydration of the tissue sections by microwaving for 10 min in 10 mM citrate buffer. Sections were cooled to room temperature, treated with 3 % hydrogen peroxide in methanol for 10 min, and blocked with 6 % horse serum for 30 min at room temperature in a humidity chamber. The sections were then incubated with primary antibody against PPARγ (diluted 1: 200; Novus, Littleton, CO, USA) or p-AMPK (diluted 1: 150; Cell Signaling) at 4 °C overnight in a humidity chamber. The sections were washed in PBS and incubated with secondary antibody (biotinylated goat anti-rabbit antibody; diluted 1: 150; Vector Laboratories, Burlingame, CA, USA) for 30 min in the humidity chamber. After further washes, the antibodies were detected with the Vector ABC complex/horseradish peroxidase (HRP) kit (Vector Laboratories, Burlingame, CA, USA) and color developed with 3,3′-diaminobenzidine tetrahydrochloride. For semiquantitation, ten photomicrographs (200×) were taken with a CCD camera, avoiding gross necrotic areas.

We analysed the expression of cardiac lipid metabolism proteins by the Western blot method as previously described in detail.²⁸ As severe LV hypertrophy is related to the impaired angiogenesis pathway, we also studied the expression of key proteins that regulate angiogenesis and signal hypoxia. Primary antibodies: HIF1α (ab463, Abcam; 1:500); fatty acid transporter (CD36; ab133625, Abcam; 1:1000); carnitine palmitoyltransferase 1 (CPT1; NBP1‐59576, Novus Biological; 1:1000); fatty acid‐binding protein (FABP3; ab133585, Abcam; 1:1000); acyl‐CoA dehydrogenase (ACADL; ab196655, Abcam; 1:3000); medium‐chain acyl‐CoA dehydrogenase (MCAD; ab110296, Abcam; 1:1000); 2,4‐dienoyl‐CoA reductase (DECR1; ab198848, Abcam; 1:2000). AMP‐activated protein kinase (AMPK; 2532 s, Cell Signaling; 1:1000); phosphorylated AMPK (Thr 172) (pAMPK; 2535 s, Cell Signaling; 1:500); SIRT1 (8469 s, Cell Signaling; 1:1000); PPARα (ab8934, Abcam; 1:1000); retinoid X receptor (RXRα; 3085 s, Cell; 1:1000); peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α; 20,658‐1‐AP, Proteintech; 1:1000). Targeted bands were normalized to the expression of cardiac GAPDH (sc‐32,233, Santa Cruz Biotechnology; 1:2000).