Effects of the dipeptides comprising leucine and lysine on lifespan and age-related stress in Caenorhabditis elegans

Abstract

The aging process is affected by various stressors. An increase in oxidative stress is related to the impairment of physiological functions and enhancement of glycative stress. Food-derived bioactive peptides have various physiological functions, including antioxidant activities. Dipeptides comprising Leu and Lys (LK and KL, respectively) have been isolated from foods; however, their physiological properties remain unclear. In this study, we investigated the antioxidant/antiglycation activity of dipeptides and their antiaging effects using Caenorhabditis elegans (C. elegans). Both dipeptides showed antioxidant activities against several reactive oxygen species (ROS) in vitro. In particular, the scavenging activity of LK against superoxide radicals was higher than KL did. Moreover, dipeptides suppressed advanced glycation end products (AGEs) formation in the BSA-glucose model. In the lifespan assays using wild-type C. elegans, both LK and KL significantly prolonged the mean lifespan by 20.9% and 11.7%, respectively. In addition, LK decreased intracellular ROS and superoxide radical levels in C. elegans. Blue autofluorescence, an indicator of glycation in C. elegans with age, was also suppressed by LK. These results suggest that dipeptides, notably LK, show an antiaging effect by suppressing oxidative and glycative stress. Our findings suggest that such dipeptides can be used as a novel functional food ingredient. Food-derived dipeptide Leu-Lys (LK) and Lys-Leu (KL) exert antioxidant and antiglycation activity in vitro. Treatment with LK prolonged the mean lifespan and maximum lifespan of C. elegans more than that of KL. Intracellular ROS and blue autofluorescence levels (indicator of aging) were suppressed by LK.

Keywords: Caenorhabditis elegans; antiaging; antiglycation; antioxidant activity; bioactive peptide; dipeptide.

FIGURE 1

Antioxidant activity of the dipeptides. (a) DPPH radical‐scavenging activity, (b) superoxide radical‐scavenging activity, and (c) hydroxyl radical‐scavenging activity. Data are expressed as the mean of three independent analyses and the SEM. Significant differences were determined by one‐way repeated ANOVA followed by the Tukey–Kramer multiple comparison test (a‐b, p < .05). Car, carnosine; KL, Lys−Leu; LK, Leu−Lys.

FIGURE 2

Antiglycation activity of dipeptides in BSA−glucose model. The data are expressed as the mean of three independent analyses and the SEM. Statistical analyses of the fluorescence intensity values were performed by using two‐way repeated ANOVA followed by Tukey–Kramer multiple comparison test. Different letters indicate significant differences on Day 7 (p < .05). AG, aminoguanidine hydrochloride; Car, carnosine; DW, distilled water; KL, Lys−Leu; LK, Leu−Lys.

FIGURE 3

Effect of dipeptides on the lifespan of C. elegans. The changes in survival rate are expressed as the mean of three independent analyses. Statistical analysis of the differences between the control groups was performed using the log‐rank test. *p < .05, **p < .01. Car, carnosine; DW, distilled water; KL, Lys−Leu; LK, Leu−Lys.

FIGURE 4

Dose‐dependent effect of LK treatment on the lifespan of C. elegans. The changes in survival rate are expressed as the mean of three independent analyses. Statistical analysis of the differences between the control groups was performed using the log‐rank test. **p < .01. DW, distilled water; LK, Leu−Lys.

FIGURE 5

Effects of LK treatment on the intracellular ROS and superoxide radical levels. (a) Images of H₂DFCDA in C. elegans; (b) Quantification of the fluorescence to determine intracellular ROS levels. (c) Images of DHE staining in C. elegans; (d) Quantification of the fluorescence to determine superoxide radical levels. Scale bar: 200 µm. The data are expressed as the mean of three independent analyses and the SEM. Statistical analysis of the differences between the control groups was performed using Student's t‐test. *p < .05, **p < .01. LK, Leu−Lys.

FIGURE 6

Measurement of blue autofluorescence intensity with age. (a) Images of blue autofluorescence in C. elegans; (b) Quantification of fluorescence intensity. Scale bar: 200 µm. The data are expressed as the mean of three independent analyses and the SEM. Statistical analysis of differences between the control groups was performed by using two‐way repeated ANOVA followed by Student's t‐test. *p < .05, **p < .01. LK, Leu‐Lys.

FIGURE 7

Effect of LK treatment on the lifespan of C. elegans under oxidative stress. The changes in survival rate are expressed as the mean of three independent analyses. DW, distilled water; LK, Leu−Lys.