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. 2023 Sep:155:106322.
doi: 10.1016/j.psyneuen.2023.106322. Epub 2023 Jun 14.

Cellular allostatic load is linked to increased energy expenditure and accelerated biological aging

Natalia Bobba-Alves  1 , Gabriel Sturm  2 , Jue Lin  3 , Sarah A Ware  4 , Kalpita R Karan  1 , Anna S Monzel  1 , Céline Bris  5 , Vincent Procaccio  6 , Guy Lenaers  7 , Albert Higgins-Chen  8 , Morgan Levine  9 , Steve Horvath  9 , Balaji S Santhanam  10 , Brett A Kaufman  4 , Michio Hirano  11 , Elissa Epel  12 , Martin Picard  13
Affiliations

Cellular allostatic load is linked to increased energy expenditure and accelerated biological aging

Natalia Bobba-Alves et al. Psychoneuroendocrinology. 2023 Sep.

Abstract

Stress triggers anticipatory physiological responses that promote survival, a phenomenon termed allostasis. However, the chronic activation of energy-dependent allostatic responses results in allostatic load, a dysregulated state that predicts functional decline, accelerates aging, and increases mortality in humans. The energetic cost and cellular basis for the damaging effects of allostatic load have not been defined. Here, by longitudinally profiling three unrelated primary human fibroblast lines across their lifespan, we find that chronic glucocorticoid exposure increases cellular energy expenditure by ∼60%, along with a metabolic shift from glycolysis to mitochondrial oxidative phosphorylation (OxPhos). This state of stress-induced hypermetabolism is linked to mtDNA instability, non-linearly affects age-related cytokines secretion, and accelerates cellular aging based on DNA methylation clocks, telomere shortening rate, and reduced lifespan. Pharmacologically normalizing OxPhos activity while further increasing energy expenditure exacerbates the accelerated aging phenotype, pointing to total energy expenditure as a potential driver of aging dynamics. Together, our findings define bioenergetic and multi-omic recalibrations of stress adaptation, underscoring increased energy expenditure and accelerated cellular aging as interrelated features of cellular allostatic load.

Keywords: Aging; Allostatic load; Chronic stress; Epigenetic aging; Glucocorticoid; Hypermetabolism; Mitochondria; Telomere.

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Conflict of interest statement

Declaration of Competing Interest The authors have no conflict of interest to declare.

Figures

Fig. 1.
Fig. 1.. Longitudinal cytologic effects of chronic glucocorticoid signaling in primary human fibroblasts.
(A) Study design: primary human fibroblasts derived from three healthy donors (Donors 1, 2, and 3) were cultured under standard conditions (control) or chronically treated with Dexamethasone (Dex, 100 nM) across their lifespan for up to 150–250 days, until replicative senescence. Cytologic parameters were evaluated every 5–7 days, while bioenergetics, DNA, RNA, and secreted factors parameters were evaluated every 10–20 days. (B) Representative images of untreated replicating control and Dex-treated cells from Donor 1 at 25 days of treatment. (C) Raw lifespan trajectories of cell volume. (D) To examine the effects of the chronic glucocorticoid signaling from the effects of aging, lifespan trajectories (left panel) and lifespan average effects (right panel) of Dex treatment on cell volume are expressed relative to the corresponding control time points for each donor. (E-F) Same as C-D but for the proportion of dead cells at each passage. n = 3 donors per group, n = 26–36 timepoints per donor. Lifespan average graphs are mean ± SEM, two-way ANOVA. * p < 0.05, * ** * p < 0.0001, ns: not significant.
Fig. 2.
Fig. 2.. Cellular allostatic load is associated with hypermetabolism.
(A-C) Energy expenditure trajectories across the cellular lifespan derived from Seahorse extracellular flux analyzer described in detail in Supplementary Material Fig. S1. (A) Lifespan trajectories (left panel) and lifespan average effects (right panel) of Dex treatment expressed relative to the corresponding control time points for each donor on basal glycolysis-derived ATP (JATP-Glyc), (B) OxPhos-derived ATP (JATP-OxPhos), and (C) total ATP production (JATP-Total) corrected for cell volume. n = 3 donors per group, 8–13 timepoints per donor. Lifespan average graphs are mean ± SEM, two-way ANOVA * p < 0.05, * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001, ns: not significant. JATP-Total is the algebraic sum of JATP-Glyc and JATP-OxPhos.
Fig. 3.
Fig. 3.. Cellular allostatic load involves a metabolic shift towards OxPhos.
(A) Fraction of basal energy production from JATP-Glyc (yellow) and basal JATP-OxPhos (green) across the lifespan. (B) Compensatory glycolytic capacity, expressed as the percentage of basal JATP-Total, achieved when OxPhos ATP production is inhibited with oligomycin. Basal JATP-Glyc levels are shown in dark yellow, and compensatory JATP-Glyc are shown in bright yellow. (C) Same as B but for spare OxPhos capacity, expressed as the percentage of basal JATP-OxPhos that can be achieved under uncoupled condition with FCCP. Basal JATP-OxPhos levels are shown in dark green, and spare JATP-OxPhos are shown in bright green. (D) Correlation between spare JATP-OxPhos/cell volume and basal JATP-OxPhos/cell volume (left panel) and between spare JATP-OxPhos/cell volume and basal JATP-Total/cell volume (right panel). (E) Lifespan trajectories (left panel) and lifespan average effects (right panel) of Dex treatment on coupling efficiency expressed relative to the corresponding control time points for each donor. (F) Same as C but for mtDNA copy number/cell volume. n = 3 donors per group, 8–13 timepoints per donor. Lifespan average graphs are mean ± SEM, two-way ANOVA. Correlation graphs show Spearman r and thick lines represent simple linear regression for each group. * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001, ns: not significant. JATP-Glyc: ATP production rate derived from glycolysis. JATP-OxPhos: ATP production rate derived from OxPhos. JATP-Total: algebraic sum of JATP-Glyc and JATP-OxPhos.
Fig. 4.
Fig. 4.. Cellular allostatic load involves transcriptional upregulation of OxPhos and mitochondrial biogenesis.
(A) Heatmaps showing the effect of Dex treatment on the expression of glycolytic genes, expressed as the Log2 of fold change (Log2FC) of normalized gene expression relative to the corresponding control time points for each donor. (B) Same as A but for genes encoding the subunits of complexes I, II, III, IV and V of the OxPhos system, with mitochondrial-encoded genes marked with ❖ (C-E). Same as A and B but for genes encoding proteins associated with (C) mtDNA maintenance, (D) mtDNA replication, and (E) mitochondrial biogenesis. Two key regulators of mitochondrial biogenesis, one positive (PGC-1a) and one negative (NRIP1) are highlighted, showing expression signatures consistent with both activated and un-repressed mitochondrial biogenesis. (F) Average effect of Dex treatment shown in A-E. Each datapoint represents the gene average of the Log2FC values throughout the entire lifespan of the three donors (n = 28 timepoints). Average graphs are mean ± SEM, with each gene shown as a single datapoint. One-sample t-test different than 0, * p < 0.05, * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001, ns: not significant. n = 3 donors per group, 9–10 timepoints per donor. Heatmap row annotation with individual gene names is provided in Supplementary Material Fig. S4.
Fig. 5.
Fig. 5.. Cellular allostatic load increases cell-free DNA levels.
(A) Lifespan trajectories (left panel) and lifespan average effects (right panel) of Dex treatment on cell-free mitochondrial DNA (cf-mtDNA) expressed relative to the corresponding control time points for each donor. (B) Correlation between cf-mtDNA and the proportion of dead cells at each passage. (C) Same as A but for cell-free nuclear DNA (cf-nDNA). (D) Same as B but for cf-nDNA. n = 3 donors per group, 6–10 timepoints per donor. Lifespan average graphs are mean ± SEM, two-way ANOVA. Correlation graphs show Spearman r and thick lines represent simple linear regression for each group. * p < 0.05, * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001, ns: not significant.
Fig. 6.
Fig. 6.. Cellular allostatic load alters cytokine release.
(A) Heatmaps showing the effect of Dex treatment on the secretion of age-related cytokines, expressed as the Log2 fold change (Log2FC) of cytokine concentration (pg/mL of medium), relative to the corresponding control time points for each donor. (B) Lifespan trajectories of cytokine concentration in cells treated with Dex relative to the corresponding control time point for each donor. Thin curves in soft red represents individual cytokines; thick curves in red represent the average of all cytokines evaluated; thick lines in gray represent the control level. (C) Lifespan trajectories (left panel) and average effects (right panel) of Dex treatment on TFPII (most upregulated cytokine) levels per mL of culture media expressed relative to the corresponding control time point for each donor. (D) Same as C but for IL6 (most downregulated cytokine). n = 3 donors per group, 6–10 timepoints per donor. Lifespan average graphs are mean ± SEM, two-way ANOVA. * ** p < 0.001, * ** * p < 0.0001, ns: not significant. Lifespan trajectories and average effects of Dex treatment on levels of every cytokine detected are shown in Supplementary Material Fig. S7.
Fig. 7.
Fig. 7.. Cellular allostatic load causes mtDNA instability.
(A) Long-range PCR (10 kb product) of mtDNA extracted from Donor 2 control and Dex-treated cells across lifespan, resolved by agarose gel electrophoresis. The presence of amplicons of sizes smaller than 10 kb reveal the presence of mtDNA molecules containing deletions. (B) Circos plots from mtDNA sequencing and eKLIPse analysis for mtDNA extracted from Donor 2 control and Dex-treated cells at days 26, 56 and 126. Each red line represents an individual deletion that spans the sequence contained between its two ends. The line thickness reflects the level of relative abundance or heteroplasmy for each deletion (see Supplementary Material Fig. S8 for all donors). (C) Lifespan trajectories (left panel) and average effects (right panel) of Dex treatment on the cumulative heteroplasmy of all deletions present at a particular time point, expressed relative to the corresponding control time point for each donor. (D) Lifespan trajectories of individual point mutations heteroplasmy found in control (left panels) and Dex-treated cells (right panels) of the three donors. n = 3 donors per group, 2–8 timepoints per donor. Datapoints in lifespan trajectories are connected using the Akima spline curve, but the datapoints for Donor 3 in (C), for which due to insufficient time points the spline fit was not feasible and therefore datapoints were connected through a straight line. Lifespan average graphs are mean ± SEM, two-way ANOVA. ns: not significant, N/A: not applicable.
Fig. 8.
Fig. 8.. Cells under chronic allostatic load display accelerated cellular aging.
(A) Lifespan trajectories of cumulative population doublings. (B) Hayflick limit for each donor of each group. (C) Group average early life doubling rate, inferred by linear mixed model of the population doubling trajectories within the first 50 days of treatment. (D) Telomere length across population doublings, with simple linear regressions for each donor of each group (left panel), and group average telomere shortening rate inferred by linear mixed model along the whole lifespan (right panel). (E) Epigenetic age calculated by the principal components (PC) PhenoAge epigenetic clock, with linear regressions for each donor of each group (left panel), and group average epigenetic aging rate inferred by linear mixed model along the whole lifespan (right panel). (F) Epigenetic aging rate for all the PC epigenetic clocks evaluated: PA: PhenoAge, H: Hannum, S&B: Skin and Blood, PT: PanTissue, GA: GrimAge. Detailed analysis of these epigenetic clocks is in Supplementary Material Fig. S11 (G) Percentage of dead cells (upper panels) and basal JATP-Total/cell volume (lower panels) across population doublings for Donor 1 (left panels), Donor 2 (middle panels) and Donor 3 (right panels). (H) Correlation between proportion of dead cells in every passage and Basal JATP-Total/cell volume. n = 3 donors per group; timepoints per donor: n = 26–36 in A, n = 4–14 in D-E, n = 8–13 in G-H. Bar graphs are mean ± SEM, Satterthwaite method. Correlation graphs show Spearman r and thick lines represent simple linear regression for each group. * p < 0.05, * * p < 0.01, * ** * p < 0.0001, ns: not significant.
Fig. 9.
Fig. 9.. Hypermetabolism, not the metabolic shift towards OxPhos, predicts cell death.
(A) Energetic phenotype of cells treated with Dex and Dex+mitoNUITs defined by Basal JATP-OxPhos/cell volume and Basal JATP-Glyc/cell volume across lifespan. (B) Lifespan average effects of Dex+mitoNUITs treatment on JATP-Total/cell volume. (C) Values across lifespan (left panel) and average effects (right panel) of basal JATP-Glyc (yellow) and basal JATP-OxPhos (green) expressed as percentage of basal JATP-Total. (D) Lifespan trajectories of cumulative population doublings. (E) Hayflick limit for each donor of each group. (F) Group averages of early life doubling rate, inferred by linear mixed model of the population doubling trajectories within the first 50 days of treatment. (G) Telomere length across population doublings, with simple linear regressions for each donor of each group (left panel), and group average telomere shortening rate inferred by linear mixed model along the whole lifespan (right panel). (H) Epigenetic age calculated by the principal components (PC)-adjusted PhenoAge epigenetic clock, with simple linear regressions for each donor of each group (left panel), and group average epigenetic aging rate inferred by linear mixed model along the whole lifespan (right panel). (J) Epigenetic aging rate for all the PC-adjusted epigenetic clocks evaluated: PA: Phene Age, H: Hannum, S&B: Skin and Blood, PT: Pan Tissue, GA: Grimm Age. (K) Percentage of dead cells (upper panels) and basal JATP-Total/cell volume (lower panels) across population doublings for Donor 1 (left panels), Donor 2 (middle panels) and Donor 3 (right panels). (H) Correlation between proportion of dead cells in every passage and Basal JATP-Total/cell volume. n = 3 donors per group; timepoints per donor: n = 7–11 in B-C, n = 26–36 in D, n = 4–14 in G-H, n = 8–13 in J-K. Lifespan average graphs are mean ± SEM, two-way ANOVA in B, Satterthwaite method in E, F, H, and I. Correlation graphs show Spearman r and thick lines represent simple linear regression for each group. * p < 0.05, * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001, ns: not significant. mitoNUITs: Mitochondrial nutrient uptake inhibitors. JATP-Glyc: ATP production rate derived from glycolysis. JATP-OxPhos: ATP production rate derived from OxPhos. JATP-Total: algebraic sum of JATP-Glyc and JATP-OxPhos.
Fig. 10.
Fig. 10.. Summary diagram.
Proposed model for the transduction of glucocorticoid signaling into cellular allostatic load and its interrelated cellular features, and the chronic downstream consequences of allostatic overload on cellular aging.
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