Research Paper Volume 13, Issue 1 pp 813—830

Carnitine promotes recovery from oxidative stress and extends lifespan in C. elegans

A potential carnitine transporter T08B1.1 promotes oxidative stress recovery and increases lifespan in C. elegans. (A) T08B1.1 is homologous to human carnitine transporter OCTN1. Protein sequence of T08B1.1 was used for blasting search with NCBI website with default setting for homologous sequences in human non-redundant protein sequences database. Table shows blasting results of the top 2 matched sequences. (B) T08B1.1 expression was elevated in glp-1 and daf-2 mutant worms. Age-matched glp-1 worms were raised at 25° C from L1 to L4 stage then changed to 20° C. Age-matched daf-2 worms were kept at 20° C. mRNAs were extracted at day-1 of adulthood and RT-qPCR was performed. Data from 3 experiments (each with 3 replicates) were analyzed with two-tailed, paired student’s t-test (*, PC) RNAi knocking down of T08B1.1 decreased L-carnitine content in C. elegans. Age-synchronized L1 worms were raised on NG medium supplemented with10 μM L-carnitine until day-1 adulthood. Worms were homogenized and relative L-carnitine content measured with L-carnitine Assay kit. Data from 2 experiments were analyzed with two-tailed, paired student’s t-test (ns, not significant, *PD) RNAi knocking down of T08B1.1 prevented L-carnitine from decreasing oxidative marker gst-4::gfp. Age-synchronized worms expressing gst-4::gfp were raised on NG medium supplemented with10 μM L-carnitine until day-4 adulthood. Worms were imaged for GFP levels. Shown are representative images from day-4 adulthood. (E) Quantification of day-2 and day-4 data from experiments shown in (D). Ten images from 2 experiments were quantified by ImageJ and analyzed with two-tailed, unpaired student’s t-test (***, PF) RNAi knocking down of T08B1.1 decreased glp-1 lifespan but not that of wild-type or daf-2 mutant. glp-1 worms were raised at 25° C from L1 to L4 stage and then changed to 20° C. WT and daf-2 worms were kept at 20° C. Dead and live worms were counted every 2 or 3 days starting from day-10 of adulthood. Data from 2 experiments (n>120 for each curve) were analyzed by log-rank test (Supplementary Table 3). (G) RNAi knocking down of T08B1.1 prevented L-carnitine from extending lifespan. Age-matched N2 wild-type worms were kept at 20° C throughout life. Dead and live worms were counted every 2 or 3 days starting from day-10 of adulthood. Data from 2 experiments (n>120 for each curve) were analyzed by log-rank test (Supplementary Table 4).

Figure 5. A potential carnitine transporter T08B1.1 promotes oxidative stress recovery and increases lifespan in C. elegans. (A) T08B1.1 is homologous to human carnitine transporter OCTN1. Protein sequence of T08B1.1 was used for blasting search with NCBI website with default setting for homologous sequences in human non-redundant protein sequences database. Table shows blasting results of the top 2 matched sequences. (B) T08B1.1 expression was elevated in glp-1 and daf-2 mutant worms. Age-matched glp-1 worms were raised at 25° C from L1 to L4 stage then changed to 20° C. Age-matched daf-2 worms were kept at 20° C. mRNAs were extracted at day-1 of adulthood and RT-qPCR was performed. Data from 3 experiments (each with 3 replicates) were analyzed with two-tailed, paired student’s t-test (*, P<0.05. **, P<0.01). Error bars indicate standard deviation of the mean. (C) RNAi knocking down of T08B1.1 decreased L-carnitine content in C. elegans. Age-synchronized L1 worms were raised on NG medium supplemented with10 μM L-carnitine until day-1 adulthood. Worms were homogenized and relative L-carnitine content measured with L-carnitine Assay kit. Data from 2 experiments were analyzed with two-tailed, paired student’s t-test (ns, not significant, *P<0.05, **P<0.01). Error bars indicate standard deviation of the mean. (D) RNAi knocking down of T08B1.1 prevented L-carnitine from decreasing oxidative marker gst-4::gfp. Age-synchronized worms expressing gst-4::gfp were raised on NG medium supplemented with10 μM L-carnitine until day-4 adulthood. Worms were imaged for GFP levels. Shown are representative images from day-4 adulthood. (E) Quantification of day-2 and day-4 data from experiments shown in (D). Ten images from 2 experiments were quantified by ImageJ and analyzed with two-tailed, unpaired student’s t-test (***, P<0.001). Error bars indicate standard deviation of the mean. (F) RNAi knocking down of T08B1.1 decreased glp-1 lifespan but not that of wild-type or daf-2 mutant. glp-1 worms were raised at 25° C from L1 to L4 stage and then changed to 20° C. WT and daf-2 worms were kept at 20° C. Dead and live worms were counted every 2 or 3 days starting from day-10 of adulthood. Data from 2 experiments (n>120 for each curve) were analyzed by log-rank test (Supplementary Table 3). (G) RNAi knocking down of T08B1.1 prevented L-carnitine from extending lifespan. Age-matched N2 wild-type worms were kept at 20° C throughout life. Dead and live worms were counted every 2 or 3 days starting from day-10 of adulthood. Data from 2 experiments (n>120 for each curve) were analyzed by log-rank test (Supplementary Table 4).