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. 2021 Feb 8;10(2):262.
doi: 10.3390/antiox10020262.

Rebaudioside A Enhances Resistance to Oxidative Stress and Extends Lifespan and Healthspan in Caenorhabditis elegans

Pan Li  1   2 , Zehua Wang  1   2 , Sin Man Lam  1   3 , Guanghou Shui  1   2
Affiliations

Rebaudioside A Enhances Resistance to Oxidative Stress and Extends Lifespan and Healthspan in Caenorhabditis elegans

Pan Li et al. Antioxidants (Basel). .

Abstract

Non-nutritive sweeteners are widely used in food and medicines to reduce energy content without compromising flavor. Herein, we report that Rebaudioside A (Reb A), a natural, non-nutritive sweetener, can extend both the lifespan and healthspan of C. elegans. The beneficial effects of Reb A were principally mediated via reducing the level of cellular reactive oxygen species (ROS) in response to oxidative stress and attenuating neutral lipid accumulation with aging. Transcriptomics analysis presented maximum differential expression of genes along the target of rapamycin (TOR) signaling pathway, which was further confirmed by quantitative real-time PCR (qPCR); while lipidomics uncovered concomitant reductions in the levels of phosphatidic acids (PAs), phosphatidylinositols (PIs) and lysophosphatidylcholines (LPCs) in worms treated with Reb A. Our results suggest that Reb A attenuates aging by acting as effective cellular antioxidants and also in lowering the ectopic accumulation of neutral lipids.

Keywords: C. elegans; TOR; aging; lipidomics; non-nutritive sweetener; oxidative stress resistance; rebaudioside A.

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Conflict of interest statement

Sin Man Lam is an employee of LipidALL Technologies.

Figures

Figure 1
Figure 1
Reb A extends lifespan in C. elegans. (A) Pumping rate in worms on the 5th and 10th day of adulthood in wild-type N2 strain on both control nematodes growth media (NGM) and NGM supplemented with rebaudioside A (Reb A) at designated concentrations (0.0083, 0.017, 0.033, and 0.05 mM). One-way ANOVA was used for multiple group comparisons and pairwise significance was evaluated by Dunnet’s test; (B) Lipofuscin fluorescence in worms on the 8th day of adulthood grown under control NGM and 0.0083 mM Reb A NGM. Statistical significance was calculated by unpaired t-test; (C) Survival plot and lifespan assay comparing worms grown on control NGM and that supplemented with 0.0083 mM Reb A. Statistical significance was calculated by log-rank (Mantel–Cox) test, * p < 0.05, *** p < 0.001. Maximum and medium survival were shown in the table appended below.
Figure 2
Figure 2
Reb A extends healthspan in C. elegans. (A) Swimming movement in C. elegans on the 5th and the 10th day of adulthood on control NGM and NGM supplemented with 0.0083 mM Reb A; (B) Osmotic avoidance behavior in C. elegans on the 1st day of adulthood on control NGM and NGM supplemented with 0.0083 mM Reb A. Statistical significance was calculated by unpaired t-test; (C) Body length and width of C. elegans on the 1st day of adulthood grown on control NGM and NGM supplemented with different concentrations of Reb A. Statistical significance was calculated by Dunnet’s test; (D) A comparison on total brood size over a seven-day reproductive period.
Figure 3
Figure 3
Reb A enhances resistance to oxidative stress in C. elegans. (A) Heat stress survival in the 6-day-old adult worms from Reb A and control groups; (B) Acute oxidative stress (treatment with 200 mM paraquat for 2 h) survival in 6-day-old adult worms from Reb A and control groups; (C) Relative cellular ROS level after treatment with 7 mM paraquat in the 6-day-old adult worms from Reb A and control groups; (D) MDA content in the 6-day-old adult worms from Reb A and control groups following oxidative stress exposure; (E) Under normal condition, basal respiration, maximal respiration, and spare capacity of 0.0083 mM Reb A-treated 6-day-old adults were not significantly different from those of the control group (n = 16 wells); (F) OCR was not statistically different between the 6-day-old adult worms treated with 0.0083 mM Reb A and the control group; (G) The 6-day-old adult worms from the 0.0083 mM Reb A group showed increased basal respiration and maximal respiration after 2 h of 7 mM paraquat treatment as compared with the control group (n = 12 wells), * p < 0.0021, ** p < 0.0332; (H) OCR under oxidative stress was significantly increased in 6-day-old adult worms treated with 0.0083 mM Reb A as compared with the control group. Statistical significance was calculated by unpaired t-test.
Figure 4
Figure 4
Reb A inhibited CeTOR signaling pathway. (A) Food intake showed no significant difference in worms grown under control NGM and NGM supplemented with both 0.0042 mM and 0.0083 mM Reb A; (B) Bidirectional clustering heat map of differentially expressed genes (DEGs) expression based on FPKM data of RNA-seq transcriptome; (C) Volcano plot illustrates DEGs in Reb A treatment groups relative to the control group. Red dots are upregulated genes while green dots were downregulated genes; (D) Top 20 GO terms based on transcriptome of Reb A-treated worms relative to controls. The vertical axis corresponds to the GO terms divided into different categories, and the horizontal axis displays the value of -log10 (p-value). The number on the right of each GO term indicates the number of DEGs. Green represents biological processes, red represents cellular components, and blue represents molecular functions; (E) Top altered pathways with Reb A treatment in C. elegans. The vertical axis corresponds to the KEGG pathways, and the horizontal axis displays the enriched value expressed as the ratio of DEG to the total gene number in each pathway. The size and color of dots indicates the DEG gene number and the p-value, respectively; (F) Expression level of genes in CeTOR signaling pathway, which include daf-15, ife-1, clk-2, rheb-1, let-363, mlst-8, and rps-6; (G) Gene expression levels in PI3K/Akt signaling pathway, and selected genes include age-1, pdk-1, akt-2, daf-18, aap-1, akt-1, and ist-1; (H) The expression levels of genes related to longevity signaling pathways of nematode include gcs-1, sod-3, sod-2, pha-4, lgg-1, fat-5, mtl-1, ctl-3, unc-51, daf-16, ctl-2, fat-6, and lips-17. Unpaired two-tailed test was used to examination of statistical significance, p value represented by GP type, *** p < 0.0002, ** p < 0.0021, * p < 0.0332, and others showed precise p values.
Figure 5
Figure 5
Reb A supplementation lowers lipid storage in C. elegans. (A) Nile red staining in 5-day-old adults from the 0.0083 mM Reb A and control groups; (B) Nile red staining in 10-day-old adults from the 0.0083 mM Reb A treatment and control groups. Statistical significance was calculated by unpaired two-tailed t test; (C) Distribution of lipid droplet diameters in 1st xdEx1001 strain in control group; (D) Distribution of lipid droplet diameter of 1st xdEx1001 strain in 0.0083 mM Reb A treatment group; (E) The adult lipid droplet diameter distribution of 1st xdEx1001 strain in 0.033 mM Reb A treatment group; (F) Quantification of the size of PLIN1:GFP rings in Reb A treatment groups relative to the control group. Statistical significance was calculated by ANOVA with post hoc Dunnet’s test.
Figure 6
Figure 6
Reb A affects alters lipid metabolism of C. elegans. (A) The content of phosphatidylcholines (PGs), phosphatidic acids (PAs), cardiolipins (CLs), lysophosphatidylcholines (LPCs), LPS, lysophospholipinositols (LPIs), phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), lysophosphatidylethanolamines (LPEs), and phosphatidylinositiols (PIs) in the 10-day-old adults were analyzed by lipidomics, presented as molar fractions normalized to total polar lipids (MFP); (B) Profiles of LPCs in 10-day-old adult worms; (C) Profiles of PAs in 10-day-old adult worms; (D) Profiles of PIs in 10-day-old adult worms. Statistical significance was calculated by unpaired two-tailed t test. ** p < 0.001 and * p < 0.05.
Figure 6
Figure 6
Reb A affects alters lipid metabolism of C. elegans. (A) The content of phosphatidylcholines (PGs), phosphatidic acids (PAs), cardiolipins (CLs), lysophosphatidylcholines (LPCs), LPS, lysophospholipinositols (LPIs), phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), lysophosphatidylethanolamines (LPEs), and phosphatidylinositiols (PIs) in the 10-day-old adults were analyzed by lipidomics, presented as molar fractions normalized to total polar lipids (MFP); (B) Profiles of LPCs in 10-day-old adult worms; (C) Profiles of PAs in 10-day-old adult worms; (D) Profiles of PIs in 10-day-old adult worms. Statistical significance was calculated by unpaired two-tailed t test. ** p < 0.001 and * p < 0.05.
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