Fig. 1: Serum from CR-treated mice can activate AMPK in cells and in mice.

a–d, Serum from CR-treated mice can activate AMPK in cells cultured in normal medium. MEFs (a), HEK293T cells (b), primary hepatocytes (c) and primary myocytes (d) were cultured in DMEM (a,b), William’s medium E (c) or Ham’s F-10 medium (d). However, FBS (a,b,d) or BSA (c) supplemented in the culture medium was replaced with an equal volume of serum from mice subjected to CR for 4 months (collected at 17:00, immediately before the next food supply; this CR serum was used hereafter, unless stated otherwise) or serum from mice on an ad libitum diet, for 4 h (AL serum; control). Cells were then lysed, followed by determination of AMPK activation by immunoblotting (IB) for pAMPKα and pACC levels. e,f, Serum from CR-treated mice activates AMPK in the muscle and liver of mice perfused with this serum. Ad libitum-fed mice were perfused through the jugular with 100 μl of CR serum or AL serum (as control), followed by determination of AMPK activation in liver (e) and muscle tissues (f) at 2 h after perfusion by IB for pAMPKα and pACC. g–i, Heat-stable, polar metabolites with low molecular weights in CR serum mediate AMPK activation. Schematic of experiment (g). MEFs were treated with CR serum as in a, except that the serum was heat-inactivated, dialysed or passed through a Lipidex column, followed by determination of AMPK activation by IB (h,i). Experiments were performed three (c–i) or five (a,b) times. Artwork in g was reproduced from Servier Medical Art under a Creative Commons Attribution 3.0 unported licence.