A microbiota-derived metabolite, 3-phenyllactic acid, prolongs healthspan by enhancing mitochondrial function and stress resilience via SKN-1/ATFS-1 in C. elegans

Abstract

The mechanisms underlying the impact of probiotic supplementation on health remain largely elusive. While previous studies primarily focus on the discovery of novel bioactive bacteria and alterations in the microbiome environment to explain potential probiotic effects, our research delves into the role of living Lactiplantibacillus (formerly known as Lactobacillus) and their conditioned media, highlighting that only the former, not dead bacteria, enhance the healthspan of Caenorhabditis elegans (C. elegans). To elucidate the underlying mechanisms, we conduct transcriptomic profiling through RNA-seq analysis in C. elegans exposed to GTB1, a strain of Lactiplantibacillus plantarum or 3-phenyllactic acid (PLA), mimicking the presence of key candidate metabolites of GTB1 and evaluating healthspan. Our findings reveal that PLA treatment significantly extends the healthspan of C. elegans by promoting energy metabolism and stress resilience in a SKN-1/ATFS-1-dependent manner. Moreover, PLA-mediated longevity is associated with a novel age-related parameter, the Healthy Aging Index (HAI), introduced in this study, which comprises healthspan-related factors such as motility, oxygen consumption rate (OCR), and ATP levels. Extending the relevance of our work to humans, we observe an inverse correlation between blood PLA levels and physical performance in patients with sarcopenia, when compared to age-matched non-sarcopenic controls. Our investigation thus sheds light on the pivotal role of the metabolite PLA in probiotics-mediated enhancement of organismal healthspan, and also hints at its potential involvement in age-associated sarcopenia. These findings warrant further investigation to delineate PLA's role in mitigating age-related declines in healthspan and resilience to external stressors.

Fig. 1. Lactiplantibacillus (GTB1) and its conditioned media improve fitness and healthspan.

a Effects of GTB1 (10 × 10⁹–10 × 10¹¹ bacteria/ml) versus the vehicle (S-basal) (black) on lifespan (p-value listed, respectively, log-rank test); colour coding assigned to all subsequent panels. b Effects of GTB1 dead cell (10 × 10⁹–10 × 10¹¹ bacteria/ml) versus the vehicle (S-basal) (black) on lifespan (p-value listed, respectively, log-rank test). c Effects of GTB1 conditioned media (25–100% in total media) versus the vehicle (0% CM) (black) on lifespan (p-value listed, respectively, log-rank test). d–i Effects of CM versus the 0% vehicle in animals regarding d lipofuscin (***p < 0.001 and ****p < 0.0001 versus the vehicle group, one-way ANOVA, n = 20 worms × three assays each), e average speed (****p < 0.0001 versus the vehicle group, one-way ANOVA, n = 10–12 worms × 5 assays each), f average pumping (***p < 0.001 and ****p < 0.0001 versus the vehicle group, one-way ANOVA, n = 30 worms × three assays each), g triglyceride (TG) content (**p = 0.002, ***p < 0.001, and ****p < 0.0001 versus the vehicle group, one-way ANOVA, n = 3 worm pellets), h thermotolerance (p = 0.169, **p = 0.008, and ***p < 0.001 versus the vehicle group, one-way ANOVA, n = 18–28 worms × 6 measurements each), i oxidative stress resistance (p = 0.914, **p = 0.004, and **p = 0.002 versus the vehicle group, one-way ANOVA, n = 20 worms × nine measurements each). Lifespan assay data are represented in Supplementary Data 1. Error bars represent the mean ± s.d.

Fig. 2. GTB1-derived metabolite 3-phenyllactic acid (PLA) promotes healthy aging in C. elegans.

a Metabolite profiling of the control, GTB1 or heat-treated GTB1-fed worms using LC-TOF/MS. b PLA (***p < 0.001 versus the vehicle group, one-way ANOVA, n = 3 worm pellets each) was selected as increased metabolite of GTB1-fed worms compared to control (OP50) or heat-inactivated- GTB1 condition (****p < 0.0001 versus the vehicle group, one-way ANOVA, n = 3 worm pellets each). c Effects of PLA versus vehicle (water) on lifespan (p-value listed, respectively, log-rank test). d–i Effects of PLA versus vehicle (water) for d lipofuscin (****p < 0.0001 versus the control group, one-way ANOVA, n = 20 worms × three assays each), e average speed (****p < 0.0001, Student’s t-test, n = 10−12 worms × 3 assays each), f average pumping (***p < 0.001 versus the vehicle group, one-way ANOVA, n = 30 worms × three assays each), g triglyceride (TG) content (****p < 0.0001 Student’s t-test, n = 3 worm pellets), h thermotolerance (****p < 0.0001 versus the control group, one-way ANOVA, n = 18–28 worms × 6 measurements each), and i oxidative stress resistance (***p < 0.001 and ****p < 0.0001 two-tailed t-test with FDR < 0.05, n = 20 worms × 9 measurements each). Lifespan assay data are represented in Supplementary Data S1. Error bars represent the mean ± s.d.

Fig. 3. PLA supplementation enhances HAI and mitochondrial oxidative phosphorylation complex activity.

Age-related comparison of longevity compound PLA (10 mM), Metformin (100 μM), Resveratrol (100 μM), LY294002 (100 μM), Rapamycin (10 μM), Kahalalide F (0.5 μM), or Lutein (100 μM) for a average speed, b oxygen consumption rate (OCR), and c ATP contents. d Age-associated HAI and e lifespan changes. Healthy Aging Index (HAI) was estimated by the f following equation. g Organismal ATP level under PLA treatment (***p < 0.001 and ****p < 0.0001 versus control worms, n = 3 worm pellets). Mitochondrial activity was examined by h, mitochondrial mass with MitoTracker green (*p < 0.05, **p < 0.01, and ****p < 0.0001 versus the control group, one-way ANOVA, n = 20 worms × nine measurements each) and i mitochondrial membrane potential with TMRE (****p < 0.0001 versus the control group, one-way ANOVA, n = 20 worms × nine measurements each). j The oxygen consumption rate examined in PLA treated condition (****p < 0.0001 versus control worms, n = 3 worm pellets). k, I The enzyme activity of mitochondrial respiratory chain complex (MRCC) k II (**p = 0.018 and ****p < 0.0001 versus control worms, n = 3 worm pellets) or l V was evaluated in worms (****p < 0.0001 versus control worms, n = 3 worm pellets). Error bars represent the mean ± s.d.

Fig. 4. PLA supplementation activate stress-response transcription factors SKN-1 and ATFS-1.

a Comparing the transcriptomes of the PLA-treated worm with those treated with either skn-1 RNAi or atfs-1 RNAi via scatter plots and Venn diagrams suggests that SKN-1 and AFTS-1 are crucial players generating the effect of PLA supplementation at the transcriptional level. b Hypothesis of PLA-induced longevity. PLA elevates organismal HAI with SKN-1/ATFS-1-mediated mitochondrial activation and stress resilience.

Fig. 5. PLA mediates SKN-1 and ATFS-1 pathway.

a Lifespan analysis of skn-1(zj15) with PLA treatment was performed. b Nuclear translocation of SKN-1::GFP was visualized using skn-1::gfp worms with PLA supplementation. Scale bars, 200 μm. c The quantification of SKN-1::GFP localization with PLA treatment (*p < 0.05 and ***p < 0.001 versus the control group, n = 20 worms × six measurements each). d Lifespan assay of atfs-1(gk3094) under PLA treatment. e ATFS-1::GFP localization was depicted with atfs-1::gfp worms under PLA treatment. Scale bars, 200 μm. f ATFS-1::GFP localization was quantified (*p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group, n = 20 worms × six measurements each). g Organismal ATP level with PLA was estimated in atfs-1(gk3094) or skn-1(zj15) deletion mutant (**p < 0.01 versus the control group, n = 20 worms × 3 measurements each). h Thermotolerance assay of N2, atfs-1(gk3094) or skn-1(zj15) mutant worms under treatment of PLA (***p < 0.001 and ****p < 0.0001 versus the control group, n = 18–28 worms × 6 measurements each, respectively). i Oxidative stress resistance of N2, atfs-1(gk3094) or skn-1(zj15) mutant animals with PLA supplementation (***p < 0.001 and ****p < 0.0001 versus the control group, n = 20 worms × 9 measurements each). Overall differences between vehicle control (water) with conditions were analysed by two-way ANOVA. Differences in individual values or between two groups were determined using two-tailed t-tests (95% confidence interval). Error bars represent the mean ± s.d.

Fig. 6. The correlation of blood PLA concentration and physical performance with human patients with sarcopenic phenotype.

a Non-sarcopenic (69.5 ± 5.89 years) or patients with sarcopnic phenotype (71.3 ± 6.62 years) blood samples were collected and D-PLA concentration in human blood samples was measured. Created in BioRender. Ryu, D. (2024) BioRender.com/s00v719. b Muscle mass and Skeletal muscle mass index of non-sarcopenic or sarcopenic participants. Muscle function of participants were evaluated with Grip strength (****p < 0.0001), Gait speed (****p < 0.0001), FTSS (****p < 0.0001), and SPPB score (****p < 0.0001).