Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger transcription profile

Abstract

Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the most robust interventions known to increase longevity in lower organisms. However, little is known about the molecular adaptations induced by CR in humans. Here, we report that long-term CR in humans inhibits the IGF-1/insulin pathway in skeletal muscle, a key metabolic tissue. We also demonstrate that CR induces dramatic changes of the skeletal muscle transcriptional profile that resemble those of younger individuals. Finally, in both rats and humans, CR evoked similar responses in the transcriptional profiles of skeletal muscle. This common signature consisted of three key pathways typically associated with longevity: IGF-1/insulin signaling, mitochondrial biogenesis, and inflammation. Furthermore, our data identify promising pathways for therapeutic targets to combat age-related diseases and promote health in humans.

Keywords: caloric restriction; human; insulin/IGF-1 signaling; skeletal muscle.

Fig. 1. Principal component analysis (PCA) and parametric analysis of gene-set enrichment (PAGE) were performed on microarray data from humans and rats

PCA of (A) humans on caloric restriction (CR) (blue) or ad libitum (AL)/Western diet (WD) (red), (B) those of younger subjects on a WD (green), and (C) rats. n=5 for both AL and CR-fed rats; n=10 and n=15 for humans on WD and CR, respectively; n=5 for younger individuals on WD. (D) PAGE comparing the top 100 significantly pathways up-regulated (red) or down-regulated (blue) by either the WD or CR (WD-Y=younger individuals eating Western diets (WD); WD-M=middle-aged individuals on WD).

Fig. 1. Principal component analysis (PCA) and parametric analysis of gene-set enrichment (PAGE) were performed on microarray data from humans and rats

PCA of (A) humans on caloric restriction (CR) (blue) or ad libitum (AL)/Western diet (WD) (red), (B) those of younger subjects on a WD (green), and (C) rats. n=5 for both AL and CR-fed rats; n=10 and n=15 for humans on WD and CR, respectively; n=5 for younger individuals on WD. (D) PAGE comparing the top 100 significantly pathways up-regulated (red) or down-regulated (blue) by either the WD or CR (WD-Y=younger individuals eating Western diets (WD); WD-M=middle-aged individuals on WD).

Fig. 2. Transcriptional and post-transcriptional modifications of the PI3K/AKT/FOXO pathway in human skeletal muscle by CR

(A) Transcriptional down-regulation of the PI3K/AKT/FOXO signaling pathway by CR. GH: growth hormone; IGF: insulin-like growth factor; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase B (PKB); FOXO-3A: forkhead box O3; FOXO-4: forkhead box O4; mTOR: mammalian target of rapamycin; LC3: microtubule-associated protein 1 light chain 3; AMPK: adenosine monophosphate-activated protein kinase; NFκB: nuclear factor-kappaB; SOD2: superoxide dismutase 2; DDB1: damage-specific DNA binding protein 1; FASL: fas ligand. Western blot of human skeletal muscle from individuals on a Western diet (WD) or caloric restricted (CR) diet. Immunoblot (B) and quantification (C) of western blots in panel a for p-T308 AKT and p-S473 AKT was performed using NIH ImageJ, and normalized to total AKT expression. (*p<0.01, **P<0.00003 (n=10 WD, 15 CR samples)). Bars indicate mean ± SEM.

Fig. 3. Parametric analysis of gene-set enrichment (PAGE) of microarray data from humans and rats

Columns show every (A) gene or (B) pathway significantly up-regulated (red) or down-regulated (blue) by CR. (C) The five most highly up-regulated and down-regulated genes, based on Z-ratio in CR compared to WD and AL respectively. (D) Venn diagram showing the pathway interactions between humans and rats (up-regulated and down-regulated pathways are shown in red and blue respectively). (E) Cell plot showing the most highly significant up-regulated (red) and down-regulated (blue) common pathways between humans and rats.

Fig. 3. Parametric analysis of gene-set enrichment (PAGE) of microarray data from humans and rats

Columns show every (A) gene or (B) pathway significantly up-regulated (red) or down-regulated (blue) by CR. (C) The five most highly up-regulated and down-regulated genes, based on Z-ratio in CR compared to WD and AL respectively. (D) Venn diagram showing the pathway interactions between humans and rats (up-regulated and down-regulated pathways are shown in red and blue respectively). (E) Cell plot showing the most highly significant up-regulated (red) and down-regulated (blue) common pathways between humans and rats.