Exercise training restores longevity-associated tryptophan metabolite 3-hydroxyanthranilic acid levels in middle-aged adults

Abstract

Aim: Recent pre-clinical evidence suggests that the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) and the related enzyme activity along the kynurenine metabolic pathway (KP) are associated with lifespan extension. We aimed to translate these findings into humans and expose exercise training as a potential non-pharmacological intervention to modulate this metabolic hub.

Methods: To explore whether recent pre-clinical findings might also be of relevance for humans, we analyzed the evolutionary conservation of KYNU and HAAO, the two core KP enzymes associated with 3-HAA. In a cross-sectional analysis of young-to-middle-aged adults (N = 84), we examined potential associations of serum 3-HAA and its precursor anthranilic acid with age. We then investigated whether 26 weeks of endurance exercise (increasing intensity (INC) during the intervention period (n = 17) vs. conventional moderate continuous training (CON) matched for energy expenditure (n = 17)) impacted 3-HAA levels, related metabolic ratios, and other KP metabolites.

Results: We demonstrate that the core KP enzymes associated with 3-HAA are evolutionarily conserved in humans. Serum 3-HAA and its precursor anthranilic acid were consistently associated with age in young-to-middle-aged adults. Both exercise modes tested induced an increase in 3-HAA levels of 134% (p < 0.001) and 85% (p < 0.001) compared with baseline, respectively, without a significant time*group interaction effect.

Conclusion: We translate the association between systemic 3-HAA levels and age from animal models into humans and highlight longer-term exercise training as an efficient strategy to boost systemic 3-HAA levels in middle-aged adults. Our findings open promising research avenues concerning the mediating role of 3-HAA in training adaptations, health, and longevity.

Keywords: aging; exercise; high‐intensity interval training; kynurenine pathway; tryptophan.

FIGURE 1

Evolutionary conservation of core kynurenine pathway enzymes associated with 3‐hydroxyanthranilic acid. A: Multiple sequence alignment of amino acid residues 195–374 of kynureninase (KYNU). B: Multiple sequence alignment of amino acid residues 1–175 of 3‐hydroxyanthranilic acid 3,4‐dioxygenase (HAAO). Full amino acid sequences are depicted in Supplement 2 and 3. Similarity between the sequences is color‐coded: Red box with white character = strict identity, red character = similarity in a group, blue frame = similarity across groups. Secondary structures are displayed above the aligned sequences and relative accessibility is color‐coded beneath: Blue = accessible, cyan = intermediate, white = buried, red = accessibility not predicted. Green triangles indicate residues involved in substrate cleavage as reported previously., ,

FIGURE 2

Tryptophan metabolite 3‐hydroxyanthranilic acid (3‐HAA) associates with age in young‐to‐middle‐aged adults. A: Schematic overview of the kynurenine pathway (KP) of tryptophan metabolism. B: Age‐related differences in the level of 3‐HAA, anthranilic acid (AA), and 3‐HAA/AA ratio between young (20–29 years of age, n = 30) and middle‐aged (30–60 years of age, n = 54) adults, illustrated by boxplots with 95% confidence intervals as whiskers. C: Scatter plots illustrating correlations (r: Pearson's coefficient; CI: 95% confidence interval) between age and level of 3‐HAA, AA, and 3‐HAA/AA ratio (pooled cohorts, N = 84). D: Heatmap showing correlations (Pearson's coefficient) between all KP metabolites, age (N = 84), and conventional serum inflammation markers (IL‐6: N = 47; IL‐10: N = 43). Significant correlations (p < 0.05) were indicated in bold. E: Sex‐related differences in levels of 3‐HAA, AA, and 3‐HAA/AA ratio (males: N = 40; females: N = 44) illustrated as boxplots with 97.5 and 2.5 percentile as whiskers. *p < 0.05; **p < 0.01; ***p < 0.001, based on independent t‐tests. CON = control group; INC = intervention group; IL‐6 = interleukin‐6; IL‐10 = interleukin‐10; Neopt = neopterin; Trp = tryptophan; Kyn = kynurenine; KTR = kynurenine‐to‐tryptophan ratio; KA = kynurenic acid; Qld = quinaldic acid; 3‐HK = 3‐hydroxykynurenine; XA = xanthurenic acid; AA = anthranilic acid; 3‐HAA = 3‐hydroxyanthranilic acid; QA = quinolinic acid; Pic = picolinic acid; 3‐HAA/AA ratio = 3‐hydroxyanthranilic acid/anthranilic acid ratio; QA/3‐HAA ratio = quinolinic acid/3‐hydroxyanthranilic acid ratio; Pic/3‐HAA ratio = picolinic acid/3‐hydroxyanthranilic acid ratio. yrs: Years T₀‐T₁: All participants completed 50 min continuous walking/cycling at 55% heart rate reserve (HR_R)). Randomization to CON/INC was performed after 10 weeks (T₁). CON participants continued 50 min continuous walking/cycling at 55%HR_R for 16 weeks (T₁‐T₃). INC participants completed 50 min continuous walking/cycling at 70% HR_R for 8 weeks (T₁‐T₂) and high‐intensity interval training (4 × 4 min at 95% HR_R) for 8 weeks (T₂ to T₃). KTR is given in μmol*L⁻¹ by mmol*L⁻¹. 3‐HAA/AA ratio, QA/3‐HAA ratio, and Pic/3‐HAA ratio are given in nmol*L⁻1 by nmol*L⁻¹.

FIGURE 3

Markers of inflammation and systemic tryptophan metabolite levels along the kynurenine pathway in response to 26 weeks of either increased intensity exercise (INC) or moderate‐intensity exercise (CON). A: Study design of the randomized controlled trial investigating serum KP modulating effects of a 26‐week endurance exercise training (conventional moderate‐intensity continuous training (CON, n = 17) versus increasing exercise intensity training (INC, n = 17)) at baseline (T₀), 10 weeks (T₁), 18 weeks (T₂), and 26 weeks (T₃). The first 10 weeks of the intervention period (indicated as gray zone in the figures) were implemented as exercise familiarization phase during which all participants conducted the conventional moderate‐intensity exercise training. *Energy expenditure for INC was matched to CON by adjusting the training time per session for intervention 2 (mean duration, 42 min per session) and intervention 3 (mean duration, 35 min per session). B‐R: Results for the separate markers and metabolites. Levels of 3‐HAA increase over 26 weeks of INC by 134%, while CON induces a descriptively smaller increase (85%). Repeated measures analyses of variance (ANOVA) and Bonferroni‐corrected post hoc comparisons (in case of significant ANOVA main effects) were conducted for all analytes. Sample sizes were n = 17 for INC versus n = 17 for CON for all analytes except for IL‐10 (INC: N = 12; CON: N = 15) *significant between group post hoc comparison; #significant within group post hoc comparison. *p < 0.05; # p < 0.05; ##p < 0.01; ###p < 0.001. Values are reported as mean ± standard error of the mean. CON = control group; INC = intervention group; KTR = kynurenine‐to‐tryptophan ratio; 3‐HAA/AA ratio = 3‐hydroxyanthranilic acid/anthranilic acid ratio; QA/3‐HAA ratio = quinolinic acid/3‐hydroxyanthranilic acid ratio; Pic/3‐HAA ratio = picolinic acid/3‐hydroxyanthranilic acid ratio. T₀‐T₁: All participants completed 50 min continuous walking/cycling at 55% heart rate reserve (HR_R)). Randomization to CON/INC was performed after 10 weeks (T₁). CON participants continued 50 min continuous walking/cycling at 55%HR_R for 16 weeks (T₁‐T₃). INC participants completed 50 min continuous walking/cycling at 70% HR_R for 8 weeks (T₁‐T₂) and high‐intensity interval training (4 × 4 min at 95% HR_R) for 8 weeks (T₂ to T₃). KTR is given in μmol*L⁻¹ by mmol*L⁻¹. 3‐HAA/AA ratio, QA/3‐HAA ratio, and Pic/3‐HAA ratio are given in nmol*L⁻1 by nmol*L⁻¹.