Metformin modulates the unfolded protein responses, altering lifespan and health-promoting effects in UPR-activated worms

Abstract

Metformin has been demonstrated to extend lifespan in various model organisms, and its molecular effects are observed in the cytoplasm and multiple organelles, including mitochondria. However, its association with the unfolded protein response (UPR) and its impact on stress resistance and locomotion remain uncertain. In this study, metformin was found to exert differential influences on both UPRmt and UPRer. The correlation between metformin's lifespan-mediating effect and its interaction with UPRs was also inconsistent. We identified a metformin-mediated lifespan extension in wild-type C. elegans and in UPRmt-activated tomm-22 and cco-1 RNAi worms. Metformin suppressed the UPRmt without compromising the lifespan extension observed in tomm-22 worms. Conversely, metformin did not affect the UPRmt but extended the lifespan of long-lived cco-1 RNAi worms. Furthermore, we investigated the effects of metformin on UPRer-activated nematodes. We observed that metformin exhibited a slight increase in the UPRer in mdt-15 RNAi worms and failed to induce lifespan extension. Surprisingly, metformin appeared to mediate lifespan extension in tmem-131 RNAi worms while suppressing the UPRer. Notably, the correlation between thermotolerance, oxidative stress resistance, and the lifespan effects of metformin in UPR-activated worms was inconsistent. Activation of UPRs, but not metformin treatment, enhanced the locomotor phenotype of these worms.

Fig 1. Effect of metformin on lifespan of wild-type and UPR-activated C. elegans.

Lifespan assays were conducted in N2 wild-type nematodes under different conditions. The nematodes were grown on OP50 in the presence of 25 mM or 50 mM metformin or without metformin (A). Nematodes were grown on either EV or RNAi bacteria for UPR^mt activation (tomm-22 and cco-1) or UPR^er activation (mdt-15 and tmem-131) without metformin treatment (B). The lifespan of UPR-activated nematodes in the presence of 50 mM metformin was assayed in comparison to the control, which is N2 grown on EV, as shown in (C-F). Each assay consisted of 100-200 age-synchronized worms. Statistical analysis was performed using the log-rank test. * and ^# denote statistical significance where Bonferroni’s p-value < 0.01 and < 0.05, responsively. Statistical analysis details are presented in Table 1.

Fig 2. Effect of metformin on UPRs activation.

Expression profiles of UPR^mt and UPR^er were conducted in SJ4100 zcls13[hsp-6::GFP] and SJ4005 zcls4[hsp-4::GFP] worms, accordingly. The expression of hsp-6::GFP in worms fed with EV, tomm-22, cco-1 and nuo-6 RNAi, and of hsp-4::GFP in worms fed with EV, mdt-15, tmem-131 and tag-355 RNAi is presented as shown (A). The treated nematodes were administered metformin at a concentration of 50 mM. Quantitative analysis of GFP signals is presented in (B). The assay was conducted on adult day 1 worms. (n = 5 for each experiment).* denotes p-value < 0.05.

Fig 3. Effect of metformin on thermotolerance in wild-type and UPR-activated C. elegans.

Thermotolerance assays were performed on N2 wild-type worms fed with EV (A), tomm-22 RNAi (B), and mdt-15 RNAi (C). Worms were exposed to metformin for their entire life (0 mM and 50 mM, as indicated). The assay was conducted every 3 days. (n = 30 for each experiment). * denotes p-value < 0.05. Statistical analysis details are presented in Table 2.

Fig 4. Effect of metformin on oxidative stress resistance in wild-type and UPR-activated C. elegans.

Oxidative stress assays were performed on N2 wild-type worms fed with EV (A), tomm-22 RNAi (B), and mdt-15 RNAi (C). Worms were treated with metformin for their entire life (0 mM and 50 mM, as indicated). The assay was conducted every 3 days. (n = 30 for each experiment). ** denotes p-value < 0.01. Statistical analysis details are presented in Table 3.

Fig 5. Effect of metformin on locomotion in wild-type and UPR-activated C. elegans.

Body bending (A-D) and movement velocity (E-H) assays were performed on N2 wild-type worms fed with EV, tomm-22 RNAi, and mdt-15 RNAi. Worms were treated with metformin for their entire life (0 mM and 50 mM, as indicated). The assay was conducted every 3 days. Each data point represents 3 independent cultures (n = 10-20). * denotes p < 0.05.