The pex5Δ mutation shortens chronological life span (CLS), alters cell morphology and remodels lipid metabolism in CR yeast. (A) Outline of subcellular localization of the Fox1p, Fox2p and Fox3p enzymes of fatty acid ß-oxidation in WT and pex5Δ cells. (B - F) Survival and the mean life spans of chronologically aging WT and pex5Δ yeast cultured in medium initially containing 0.2%, 0.5%, 1% or 2% glucose. Data are presented as means ± SEM (n = 16-38; ***p < 0.001; **p < 0.01). (G - I) Levels of free fatty acids (FFA), diacylglycerols (DAG) and triacylglycerols (TAG) in WT and pex5Δ cells grown on 0.2% glucose and taken for analyses at the indicated time-points. FFA and TAG were measured by quantitative mass spectrometry. The levels of DAG were quantitated by densitometric analysis of TLC plates. Data are presented as means ± SEM (n = 3-8; ***p < 0.001; **p < 0.01; *p < 0.05). (J and K) Transmission electron micrographs (J) and spectra of lipids extracted from purified endoplasmic reticulum (ER) and lipid bodies (LB) and analyzed by TLC (K) for WT and pex5Δ (Δ5) yeast grown on 0.2% glucose and taken for analyses at the indicated time-points. Abbreviations: Cta1p, peroxisomal catalase; D, diauxic growth phase; EE, ethyl esters; ERG, ergosterol; FA-CoA, CoA esters of fatty acids; L, logarithmic growth phase; M, mitochondrion; N, nucleus; PD, post-diauxic growth phase; ST, stationary growth phase.

(A) The spectra of proteins recovered in total cell lysates, purified ER and mitochondria of WT and pex5Δ cells that were grown under CR on 0.2% glucose and taken for analyses at the indicated time-points. (B) Functional categories of proteins that were enriched or depleted in the total cell lysate, ER and mitochondria of pex5Δ cells (as compared to WT cells) under CR conditions. Lack of 91 of these proteins increased the CLS of yeast under CR, suggesting their essential pro-aging role in longevity regulation when calorie supply is limited.

(A) A microplate assay for measuring yeast CLS by monitoring optical density at 600 nm (OD₆₀₀) was used for screening representative compounds from several commercial libraries for small molecules that extend the CLS of pex5Δ cells grown under CR on 0.5% glucose. (B) The OD₆₀₀ of a cell culture in the replica microplate following incubation for 16 to 24 hours correlates with the number of viable cells present in this culture before it was taken from the master microplate for replica plating. (C) The effect of various concentrations of the identified anti-aging small molecules on the CLS of the pex5Δ (Δ) strain under CR conditions. The "OD₆₀₀ at day 14/OD₆₀₀ at day 1" ratio was used as a measure of CLS. Data are presented as means ± SEM (n = 3-5; ***p < 0.001; **p < 0.01; *p < 0.05). The anti-aging small molecules LCA, AS, BT, NF and ID belong to five chemical groups.

(A - D) Effect of various bile acids on survival (A) and on the mean and maximum life spans (B - D) of chronologically aging WT strain grown under CR conditions on 0.2% glucose. Data are presented as means ± SEM (n = 3-28; ***p < 0.001; **p < 0.01; *p < 0.05). (E) Structure and hydrophilic/hydrophobic properties of bile acids. The R1 (α), R3 (α) and R5 (α) hydroxyl groups at the positions 6, 7 and 12 in the six-member rings B and C of the steroid nucleus increase polarity of the hydrophilic (concave) side [α-face] of the nucleus by being located below the nucleus and axially to its plane. The R4 (β) hydroxyl group at the position 7 in the six-member ring B of the steroid nucleus confers polarity of the hydrophobic (convex) side [β-face] of the nucleus by being located above the nucleus and equatorially to its plane.

(A - F) Effect of various concentrations of LCA on survival (A - D) and on the fold increase in the mean (E) or maximum (F) life span of chronologically aging WT strain cultured in medium initially containing 0.2%, 0.5%, 1% or 2% glucose. Data are presented as means ± SEM (n = 3-28). (G - I) Effect of 50 μM LCA on the mean or maximum CLS of WT yeast cultured in medium initially containing 0.2%, 0.5%, 1% or 2% glucose. Data are presented as means ± SEM (n = 12-28; ***p < 0.001).

(A - C) Levels of triacylglycerols (TAG) and free fatty acids (FFA) measured by quantitative mass spectrometry (A and B, respectively) and of diacylglycerols (DAG) monitored by TLC (C) in WT cells grown in medium with or without LCA. (D - F) Viability of WT cells pre-grown in medium with or without LCA and then treated for 2 h with palmitoleic acid (D), oleic acid (E) or DiC8 diacylglycerol (F). (G - I) Percent of WT cells (pre-grown in medium with or without LCA) that following their treatment with palmitoleic acid (G), oleic acid (H) or DiC8 diacylglycerol (I) displayed Annexin V negative and PI positive (Annexin V^- and PI⁺) staining characteristic of necrotic cell death. Data are presented as means ± SEM (n = 3-9; ***p < 0.001; **p < 0.01). WT cells grown on 0.2% glucose in the presence or absence of LCA were taken for analyses at day 7, when they reached reproductive maturation by entering into ST phase.

(A) Percent of WT cells grown in medium with or without LCA and exhibiting a tubular mitochondrial network or fragmented mitochondria. Mitochondria were visualized by indirect immunofluorescence microscopy using monoclonal anti-porin primary antibodies and Alexa Fluor 568-conjugated goat anti-mouse IgG secondary antibodies. (B - D) Oxygen consumption by WT cells grown in medium with or without LCA (B), their mitochondrial membrane potential ΔΨ (C) and their ROS levels (D). ΔΨ and ROS were visualized in living cells by fluorescence microscopy using fluorescent dyes Rhodamine 123 or Dihydrorhodamine 123, respectively. (E) The resistance of WT cells pre-grown in medium with or without LCA to chronic oxidative, thermal and osmotic stresses. (F) Viability of WT cells pre-grown in medium with or without LCA and then treated for 1 h with hydrogen peroxide or acetic acid (AcOH) to induce mitochondria-controlled apoptosis. (G- I) The frequencies of rho^- and rho⁰mutations in mitochondrial DNA (G), rib2 and rib3 mutations in mitochondrial DNA (H), and of can1 (Can^r) mutations in nuclear DNA (I) of WT cells grown in medium with or without LCA. Data in A - D and F - I are presented as means ± SEM (n = 4-17; ***p < 0.001; **p < 0.01). WT cells grown on 0.2% glucose in the presence or absence of LCA were taken for analyses at day 7, when they reached reproductive maturation by entering into ST phase.

(A and B) Effect of LCA on the mean (A) and maximum (B) life spans of chronologically aging WT strain. Data are presented as means ± SEM (n = 12-28; ***p < 0.001). (C - E) Effect of LCA on the fold increase in the mean (C) or maximum (D) life span of chronologically aging WT strain. Data are presented as means ± SEM (n = 12-28). Cells in A to D were cultured in medium initially containing 0.2%, 0.5%, 1% or 2% glucose in the presence of LCA (50 μM) or in its absence. Survival data are provided in Supplementary Figure 9. (E and F) Outline of pro- and anti-aging processes that are controlled by the TOR and/or cAMP/PKA signaling pathways and are modulated by LCA in WT cells grown under non-CR (E) or CR (F) conditions. Activation arrows and inhibition bars denote pro-aging processes (displayed in green color), anti-aging processes (displayed in red color) or processes whose role in longevity regulation is presently unknown (displayed in black color). Doted lines denote hypothetical processes. Abbreviations: PM, plasma membrane.

(A and B) Effect of LCA on the mean (A) and maximum (B) life spans of chronologically aging tor1Δ strain. Data are presented as means ± SEM (n = 4-7; ***p < 0.001). (C and D) Effect of LCA on the fold increase in the mean (C) or maximum (D) life spans of chronologically aging tor1Δ and WT strains. Data are presented as means ± SEM (n = 4-7). Cells in A to D were cultured in medium initially containing 0.2%, 0.5%, 1% or 2% glucose in the presence of LCA (50 μM) or in its absence. Survival data are provided in Supplementary Figure 10. (E and F) Outline of pro- and anti-aging processes that are controlled by the TOR and/or cAMP/PKA signaling pathways and are modulated by LCA in tor1Δcells grown under non-CR (E) or CR (F) conditions.

The currently accepted (A) and updated, based on this study (B), outlines of pro- and anti-aging processes are shown. Activation arrows and inhibition bars denote pro-aging processes (displayed in green color), anti-aging processes (displayed in red color) or processes whose role in longevity regulation was unknown (displayed in black color). Doted lines denote hypothetical, until this study, processes. See text for details.