Differentially expressed genes as quantified by deep sequencing analysis for (A)
daf-2 nematodes, (B) IRS1−/− MEFs and (C) IR+/− MEFs, all in comparison to respective controls; black dots indicate no differential regulation, red and green dots indicate regulation according to one statistical method only. Blue dots indicate regulation according to both statistical methods (used for further analysis). (D) functional groups of down-regulated and up-regulated genes (cut-off for at least two out of three sets: p=0.05). For details, please see also Table S1. (E) Venn analyses of differentially expressed genes derived from daf-2 nematodes, IRS1−/− MEFs and IR+/− MEFs (cut-off for all three sets: p=0.05). For details, please see also Table 2. (F) Lifespan analyses of N2 nematodes exposed to control RNAi (empty circles) and daf-2 RNAi (filled triangles) in comparison to exposure against B0513.5 RNAi (closed circles) alone and co-incubation with daf-2 RNAi (empty triangles, respectively). (G) Lifespan analysis of N2 nematodes exposed to L-proline (filled triangles) or solvent (empty circles). (H) Expression of B0513.5/prodh mRNA in the absence (white bars) and presence (black bars bars) of daf-2 RNAi for 48 h in wild-type (N2) and aak-2 mutant nematodes. (I) Oxygen consumption in N2 nematodes treated with control RNAi (white bars) or daf-2 RNAi (black bars), and the additional presence of RNAi against B0513.5/prodh (grey/striped bars), all after 48 h of treatment. (J) ROS levels in nematodes treated with RNAis as in Panel (I) for 48 h; mev-1(kn1) mutants and paraquat treatment for 1 h serve as positive controls. In panels I and J relative values are depicted; for absolute values, see Figure S4. In panels H J values are given as mean ± SD. *p<0.05, **p<0.01, ***p<0.001 versus respective controls. (K) Impaired insulin-/IGF1-signaling promotes L-proline catabolism to employ ROS as a mitochondrial second messenger culminating in extended life span: Impaired IIS causes reduction of glucose uptake in C. elegans, which leads to an intermittent energy deficit that activates mitochondrial respiration by increasing L-proline catabolism in an aak-2 dependent manner. This induction of mitochondrial respiration generates a transient ROS signal, which is sensed by PMK-1 and SKN-1 to secondarily cause an adaptive response to increase the respective activities of superoxide dismutase and catalase which ultimately terminate the initial ROS signal, in parallel leading to increased stress resistance and extended C. elegans life span.