A naturally occurring polyacetylene isolated from carrots promotes health and delays signatures of aging

The study addresses the need for effective, bioavailable phytochemicals that target fundamental aging pathways. Aging increases risk for chronic diseases such as type 2 diabetes, cancer, and neurodegeneration, often via shared mechanisms including dysregulated energy metabolism and mitochondrial dysfunction. Caloric restriction and mimetics that induce transient energy deficits can extend healthspan and lifespan across species. Key mediators include mitochondrial ATP synthase (central to ATP production) and signaling pathways such as AMPK (activated by energy stress) and NRF2 (a master regulator of antioxidant responses), which declines with age. Existing phytochemicals (resveratrol, quercetin, EGCG, curcumin) face bioavailability and stability limitations with mixed clinical efficacy. The research seeks to discover novel natural compounds that decrease ATP acutely and activate NRF2, with beneficial effects on organismal health and aging phenotypes, and to elucidate their mechanisms and translational potential.

- Mitochondrial ATP synthase inhibition can activate AMPK, eliciting adaptive responses to energy stress associated with longevity benefits. - NRF2 activation enhances antioxidant defenses and is downregulated with age; pharmacological activation mitigates aging phenotypes. - Several phytochemicals modulate these pathways but have limited bioavailability or stability and inconsistent efficacy in clinical contexts. - Prior work shows ATP synthase subunits (ATP5A/OSCP) influence longevity in C. elegans; partial downregulation extends lifespan. Piceatannol inhibits the F1 ATP synthase and shares phenotypes related to metabolic improvement and anticancer effects. - Exercise benefits and metabolic improvements often rely on ROS-mediated signaling (mitohormesis), which can be blunted by antioxidant supplementation. These insights motivate screening for a carrot-derived polyacetylene with superior potency and bioavailability that targets ATP synthase and NRF2 pathways.

- Compound screening: A library of 1,200 phytochemicals from edible plants (AnalytiCon Discovery) was screened in HepG2 cells for acute ATP reduction (15 min). Top candidates (5–10% ATP decrease) were tested for NRF2 activation via a HEK293 luciferase reporter assay. Controls included oligomycin, piceatannol, and Bz-423. - Structural elucidation and synthesis: The carrot-derived diacetylene (isofalcarintriol, IFT) was structurally confirmed by NMR (including HMBC, 1H J coupling indicating trans C10-C11). A modular asymmetric synthesis provided all E-isofalcarintriol stereoisomers; absolute configuration assigned via enantioselective SFC; enantiomeric excess >95%. Gram-scale synthesis of (3S,8R,9R)-IFT (1a) achieved. LC-MS quantification in Daucus carota roots estimated 3.8–8.9 μg/g dry weight. - Cell assays: ATP dynamics (CellTiter-Glo) measured at 15 min, 1 h, 24 h; Real-Time ATP Rate Assay to partition mitochondrial vs glycolytic ATP production; mitochondrial membrane potential (TMRE); ROS (DCF-DA) and H2O2 (Amplex Red); NRF2 reporter assays including NRF2−/− HEK293 and catalase overexpression. - Target identification: Chemical proteomics using biotinylated IFT (biotin-IFT) pulldowns in HepG2 (intact cells) and HEK293 (lysates), streptavidin capture, LC-MS/MS (Orbitrap Fusion Lumos). Proteins enriched ≥1.5× over negative control (biotin-alkene) with ≥2 peptides were considered binders. - Mitochondrial respiration: Seahorse XF Cell Mito Stress Assay in HepG2 (acute injection and overnight pre-treatment) and adapted Seahorse protocol in C. elegans; indirect calorimetry in mice. - C. elegans experiments: Lifespan assays (N2 wild-type, skn-1/NRF2-deficient, aak-2/AMPK-deficient), RNAi knockdown of atp-1 (ATP5A) and atp-3 (ATP5O); oxidative stress resistance (paraquat); neurodegeneration models (GMC101 Aβ-induced paralysis; huntingtin polyQ strains AM23 control, AM716 pathological) and motility (thrashing); mitochondrial mass (mtDNA/nDNA qPCR); ATP quantification. - Microscopy: IFT-alkyne localization via copper-catalyzed click chemistry with Azide-488 and co-staining with MitoTracker Deep Red in HepG2 (confocal imaging). - Mouse studies: C57BL/6NRj mice (male and female). High-fat diet (HFD) cohort: 0.1 mg/kg/day IFT in drinking water; toxicity study up to 2.5 mg/kg (liver enzymes). Outcomes: glucose tolerance tests (GTT), fasting glucose, body mass/composition, blood lipids, indirect calorimetry, exercise endurance (treadmill), mitochondrial mass in gastrocnemius (mtDNA/nDNA). Aged mice (chow diet): IFT started at 16 months; longitudinal frailty index (31-item), phenotypic age, treadmill endurance, grip strength (males), ECG-derived HRV (females), hematology and plasma cytokines.

- Discovery and potency: From 1,200 phytochemicals, 29 reduced ATP by 5–10%; 3 activated NRF2 (10-gingerol, alnusone, and IFT). IFT induced ~30-fold NRF2 activation, exceeding sulforaphane; no activation in NRF2−/− cells. - Structure and synthesis: Absolute configuration assigned as (3S,8R,9R,E)-heptadeca-10-en-4,6-diyne-3,8,9-triol; eSFC ee >95%; synthetic 1a and 1b activated NRF2 (26–25-fold at 10 μM), ent-forms inactive. Natural abundance in carrot root: 3.8–8.9 μg/g dry weight. - Longevity in C. elegans: IFT (1 nM) extended mean lifespan by up to 17% (also effective at 0.1 and 10 nM with lesser magnitude). No lifespan extension in skn-1/NRF2-deficient or aak-2/AMPK-deficient worms. RNAi of atp-1 or atp-3 increased lifespan; epistasis indicated dependence on ATP synthase function. Pharmacological F1 inhibitors (piceatannol, Bz-423) extended lifespan; oligomycin did not. - Target engagement and bioenergetics: Biotin-IFT pulldown enriched ATP5A (α-subunit) and ATP5O (OSCP). In HepG2, IFT acutely decreased ATP (15 min), transiently increased (~1 h), normalized by 24 h; in C. elegans ATP decreased acutely and remained elevated long-term. Real-Time ATP Rate: decreased mitochondrial ATP production with glycolysis unaffected. Membrane potential increased at 15 min, then normalized. Seahorse: IFT reduced OCR and ATP-linked respiration acutely and after overnight treatment; maximal respiration preserved acutely but reduced after overnight exposure. In C. elegans, basal OCR decreased (10 nM, p=0.038), maximal respiration unaffected. Indirect calorimetry showed reduced O2 consumption in young female mice on chow (p=0.031), similar trends in males. - Signaling and ROS: IFT induced early ROS in cells and worms (15 min), with lower ROS at later times. Catalase overexpression blunted NRF2 activation by IFT; ctl-1 overexpression suppressed lifespan extension, supporting mitohormetic ROS signaling. AMPK phosphorylation increased within 1 h in cells and worms. - Neurodegeneration models: IFT (1 nM) delayed paralysis in Aβ-expressing GMC101 worms and improved motility in huntingtin polyQ AM716 worms. - Anticancer effects: IFT selectively inhibited MCF-7 proliferation while sparing or enhancing HMEPC at 0.1–1 μM; inhibited proliferation of HepG2 and HT-29 (>1 μM). Soft agar colony formation markedly reduced: near-complete extinction in HepG2 and HT-29; significant reduction in MCF-7 colony numbers and size. - Mouse metabolic health (HFD): No adverse liver toxicity up to 2.5 mg/kg (2-week test). At 0.1 mg/kg/day, improved GTT in males (15 min p=0.01) and females (120 min p=0.02); fasting glucose reduced from week 16 (males p=0.0022; females p=0.0048). Body mass/composition and lipids unchanged. - Mitochondrial biogenesis: mtDNA/nDNA increased by 27% in HepG2 (p=0.0014), 17% in C. elegans (10 nM, p=0.0238), and ~26% in mouse gastrocnemius (both sexes, p=0.0222). - Physical performance: C. elegans motility increased (day 5 thrashing, p=0.0134). Treadmill endurance improved in HFD mice (females p=0.0318; males trend p=0.07) and in aged mice (sex-combined p=0.03). - Frailty and aging (aged mice): No lifespan extension, but lower frailty index scores across ages in males (22–31 months, multiple p-values significant) and females (p=0.03), decreased phenotypic age in males. Improved grimace and breathing scores; reduced total frailty at endpoints (p=0.01). Sex-specific benefits: males showed higher grip strength (p=0.0036); females had increased HRV (p=0.0014) and CV (p=0.002), reduced WBC (p=0.04) and lymphocytes (p=0.048), increased IL-4 (p=0.0064) and IL-10 (p=0.0070).

The findings demonstrate that isofalcarintriol, a carrot-derived polyacetylene, is a potent NRF2 activator and selective, reversible modulator of mitochondrial ATP synthase activity, principally engaging ATP5A/ATP5O. The acute, partial inhibition of ATP synthase triggers a mitohormetic ROS signal and AMPK activation, driving adaptive responses including enhanced oxidative stress resistance, mitochondrial biogenesis, and improved bioenergetics. These molecular events translate into organismal benefits: extended lifespan and improved motility in C. elegans, improved glucose handling, enhanced endurance, and reduced frailty parameters in mice. Anticancer and anti-aggregation effects in cellular and nematode models suggest broader relevance to age-related diseases such as cancer and neurodegeneration. The conserved nature of the response across species and the low bioactive concentrations support the translational potential of IFT as an exercise-mimetic and metabolic modulator targeting core aging hallmarks.

This work identifies and fully characterizes isofalcarintriol from carrots as a potent, bioactive phytochemical that activates NRF2 and transiently inhibits mitochondrial ATP synthase, inducing mitohormetic adaptations. Through chemical synthesis and target validation, the study links ATP synthase engagement to ROS-AMPK signaling, mitochondrial biogenesis, and improved health metrics across species. In mice, low-dose chronic supplementation improved glucose tolerance, endurance capacity, and multiple frailty parameters without overt toxicity. Isofalcarintriol emerges as a promising candidate for development as a nutritional supplement or therapeutic to ameliorate aging-related dysfunctions. Future research should clarify pharmacokinetics and bioavailability in vivo, delineate tissue-specific mechanisms (e.g., GLUT4 translocation), explore dose-response and sex-specific effects, quantify mitochondrial dynamics, and advance to preclinical disease models and clinical evaluation.

- No increase in overall lifespan observed in mice despite healthspan improvements. - Several benefits exhibited sex-specificity (e.g., grip strength in males, HRV and cytokine changes in females), requiring further validation and mechanistic understanding. - Mitochondrial morphological changes were only qualitatively observed; advanced imaging quantification is needed. - While chemical proteomics implicates ATP5A/ATP5O, in vivo target engagement and binding sites require deeper structural/biophysical confirmation. - Long-term pharmacokinetics, tissue distribution, and bioavailability of IFT in mammals were not comprehensively profiled. - Doses and delivery were limited to drinking water; alternative formulations and dosing regimens merit evaluation.