Figure 2. Autophagy Induction and Metabolic Effects of Hydroxycitrate and Other Caloric Restriction Mimetics.
(A) Inhibition of hydroxycitrate (HC)-induced autophagy by microinjection of acetyl coenzyme A (AcCoA) but not coenzyme A (CoA). U2OS cells stably expressing the autophagic marker GFP-LC3 were treated with HC for 6 hr and injected with 10 μM AcCoA or CoA. Representative pictures (left panel) and quantification (right panel, mean ± SD, n = 3).
(B) Deacetylation of cytoplasmic proteins in response to 20 mM HC or cultured in a nutrient-free condition (NF). Representative pictures (left panel) and quantification (right panel, mean ± SEM, n = 3).
(C) LC3I to LC3II conversion induced by starvation (48 hr) or short-term intraperitoneal injection of HC, C646, resveratrol (Resv), spermidine (Spd), or rapamycin (Rapa) in liver. For the characterization of the mode of action of HC, see Figure S1.
(D) Weight loss (mean ± SD, n = 5) induced by 24 and 48 hr starvation or administration of the indicated agents in C57BL/6 mice.
(E) Heatmap depicting log2 fold changes to the control of metabolite signals found significantly altered in the plasma of mice after 48 hr starvation or after two injections of corresponding caloric restriction mimetics (CRMs). For other organs, see Figure S2. For the complete list of metabolites, see Table S3.
(F) Summary of significant (p < 0.05) metabolic alterations elicited by 48 hr of starvation or CRMs in different mouse tissues and plasma of data shown in (E).CRMs-induced alterations were considered as convergent with starvation when they had the same sign.
(G) Effect of starvation and CRMs on plasma levels of insulin growth factor 1 (IGF-1) and IGF binding protein 1 (IGFBP1). Results are depicted as mean ± SEM (n = 3, two experiments).
Statistical analysis was performed by Student's t test in comparison with the control condition (B, D, and G) and by Fisher's exact test to compare convergence incidences with those in NF (F). ***p < 0.001, **p < 0.01, *p < 0.05; ns, not significant.