APPA Increases Lifespan and Stress Resistance via Lipid Metabolism and Insulin/IGF-1 Signal Pathway in Caenorhabditis elegans

Abstract

Animal studies have proven that 1-acetyl-5-phenyl-1H-pyrrol-3-yl acetate (APPA) is a powerful antioxidant as a novel aldose reductase inhibitor independently synthesized by our laboratory; however, there is no current information on APPA's anti-aging mechanism. Therefore, this study examined the impact and mechanism of APPA's anti-aging and anti-oxidation capacity using the Caenorhabditis elegans model. The results demonstrated that APPA increases C. elegans' longevity without affecting the typical metabolism of Escherichia coli OP50 (OP50). APPA also had a non-toxic effect on C. elegans, increased locomotor ability, decreased the levels of reactive oxygen species, lipofuscin, and fat, and increased anti-stress capacity. QRT-PCR analysis further revealed that APPA upregulated the expression of antioxidant genes, including sod-3, gst-4, and hsp-16.2, and the critical downstream transcription factors, daf-16, skn-1, and hsf-1 of the insulin/insulin-like growth factor (IGF) receptor, daf-2. In addition, fat-6 and nhr-80 were upregulated. However, the APPA's life-prolonging effects were absent on the daf-2, daf-16, skn-1, and hsf-1 mutants implying that the APPA's life-prolonging mechanism depends on the insulin/IGF-1 signaling system. The transcriptome sequencing also revealed that the mitochondrial route was also strongly associated with the APPA life extension, consistent with mev-1 and isp-1 mutant life assays. These findings aid in the investigation of APPA's longevity extension mechanism.

Keywords: APPA; Caenorhabditis elegans; insulin/IGF-1; lifespan; stress resistant.

Figure 1

Effects of APPA concentrations on nematode lifespan and growth of E. coli OP50. (A) The chemical structure of APPA. (B) Survival curve of nematodes fed 0, 0.09, 0.18, and 0.36 mg/mL APPA under normal culture conditions of 20 °C, among which APPA 0.18 mg/mL extended the average life span of nematodes by 4.92% compared with the control group. n = 3 (100 individuals per group), Kaplan–Meier survival analysis with Log-Rank test. (C) APPA did not affect the growth of OP50, which was detected by OD600. Data were analyzed by a Student’s t-test using GraphPad 7. Values are presented as the mean ± SEM.

Figure 2

Effects of APPA on lipofuscin accumulation in nematodes. (A) Blue autofluorescent lipofuscin fluorescence images in nematodes. n = 3 (30 individuals per group). (B) Fluorescence quantitation of blue autofluorescent lipofuscin in nematodes. Lipofuscin accumulation in nematodes was measured on the eighth day of administration, and APPA significantly decreased lipofuscin accumulation compared with control. The data were analyzed using a Student’s t-test. The values were shown as the mean ± SEM, ** p < 0.01.

Figure 3

Effects of APPA on fat accumulation and related genes in nematodes. (A) Compared with controls. Fat accumulation in nematodes was measured on the fifth day of administration. Oil red O staining diagram under bright field conditions. n = 3 (30 individuals per group). (B) Quantitative results of nematodes stained with oil red O. Compared with the control group, APPA significantly reduced fat levels in nematodes. (C) APPA enhances the expression of fat-6 and nhr-80. Numerical data were analyzed by Student’s t-test using GraphPad 7 and values were presented as mean ± SEM, n = 3 (2000 individuals per group) * p < 0.05.

Figure 4

APPA enhanced the antioxidant stress and heat stress resistance of nematodes. APPA enhanced the activity of SOD and CAT. APPA enhanced SOD (A) and CAT (B) activity in nematodes treated for five days. Data in bar graphs are expressed as mean ± SEM. (* p < 0.05, ** p < 0.01, two-tailed Student’s t-test). (C) Fluorescent images of ROS levels in nematodes. n = 3 (30 individuals per group). (D) Survival rate of nematodes under 35 °C heat stress for 5 h. n = 3 (30 individuals per group).*** p < 0.001 (E) Survival rate of nematodes under oxidative stress of 50 mM PQ. Numerical data were analyzed by Log-rank test and values were presented as mean ± SEM, * p < 0.05. n = 3 (30 individuals per group). (F) Fluorescence quantification of ROS in nematodes. The data were analyzed using a Log-rank t-test. The values were shown as the mean ± SEM, **** p < 0.0001.

Figure 5

Longevity mechanism mediated by APPA. (A) APPA prolonged the lifespan of eat-2 (ad1116). APPA treatment significantly increased the lifespan of eat-2 (ad1116). n = 3 (100 individuals per group). (B) Effects of APPA treatment on lifespan of daf-2 (e1370). n = 3 (100 individuals per group). (C) Effect of APPA treatment on lifespan of daf-16 (mgDf50). n = 3 (100 individuals per group). (D) Effects of APPA treatment on the lifespan of skn-1 (zu67). n = 3 (100 individuals per group). (E) Effects of APPA treatment on the lifespan of hsf-1 (sy441). n = 3 (100 individuals per group). (F) daf-16, skn-1 and hsf-1 mRNA levels in nematodes treated with APPA. n = 3 (2000 individuals per group). Statistical analysis of the lifespan was performed using GraphPad 7 and p values were calculated by the log-rank test. Numerical data were analyzed by Student’s t-test and values were presented as mean ± SEM, * p < 0.05, ** p < 0.01.

Figure 6

The APPA-mediated effect is associated with antioxidant genes. (A) Fluorescent image of CF1553. n = 3 (30 individuals per group). (B) Effect of APPA on the expression of SOD-3::GFP protein in nematodes (CF1553). APPA significantly induced expression of SOD-3::GFP. (C) The mRNA relative levels of sod-3, gst-4, and hsp-16.2, compared with control group. APPA significantly induced the expression of sod-3, gst-4 and hsp-16.2 mRNA. The images were analyzed with ImageJ software and numerical data were analyzed by Student’s t-test using GraphPad 7. n = 3 (2000 individuals per group) Values were presented as mean ± SEM, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 7

Effect of APPA on daf-16 entry into nucleus. (A) Fluorescence microscopy was used to observe the nuclear translocation of DAF-16::GFP, indicating nuclear translocation of daf-16 and cytoplasmic indicating no daf-16 nucleation. n = 3 (30 individuals per group). (B) The proportion of the total number of nematodes admitted to the nucleus. Compared with the control group, APPA significantly increased the percentage of daf-16 into the nucleus.

Figure 8

The effect of APPA on the regulation of nematodes and the mechanism by which APPA extends the lifespan of nematodes depend on the mitochondrial pathway. (A) There were 1548 upregulated genes and 2173 downregulated genes in the APPA-treated group (FDR < 0.01 and Fold Change ≥ 2). n = 3 (2000 individuals per group). (B) Effects of APPA on the lifespan of mev-1 (kn1). n = 3 (30 individuals per group). (C) Effects of APPA on the lifespan of isp-1 (qm150). n = 3 (30 individuals per group). (D) GO analysis of differential genes regulated by APPA treatment group. (E) KEGG analysis of differential genes in APPA-treated nematodes.

Figure 9

The KEGG analysis of differentially regulated genes.