Metformin Activity on Longevity and Mitochondrial Unfolded Protein Response (UPR^mt) across UPR^mt Invoked C. elegans

Abstract

Background: Metformin, a first line drug for the treatment of type 2 diabetes mellitus, has been implicated to slow down ageing in various organisms including C elegans. It has been shown that the drug tends to accumulate in mitochondria. Recently, the mitochondrial unfolded response (UPRmt) was shown to affect lifespan in UPRmt- activated worms. UPRmt is mitochondrial stress response pathway instigated in response to alleviate mitochondrial proteostasis. We, therefore, surmised that metformin might affect ageing by exerting its effect on UPRmt. The objective of this study is investigating the interaction of metformin with UPRmt on lifespan of worms. Methods: Synchronized 100 to 150 wildtype (N2) worms were laid for both metformin treated and untreated (control) group. The survival of the worms was scored everyday starting from adult day 1 by gentle touch. The UPRmt was induced by feeding the nematodes with dsRNA-producing (RNAi) bacteria including ech-6, pdhb-1, and wah-1 along with empty vector (EV) as a control. IPTG (isopropylβ-D-thiogalactoside) at 5 mM was used to activate dsRNA synthesis. Metformin at final concentration of 50 mM was supplemented onto the cultured agar. hsp-6p::GFP was used to observe UPRmt starting from day 2 of adult life. Results: Metformin significantly increased lifespan in wildtype worms fed with OP50 bacteria and EV. Lifespan of ech-6 and pdhb-1 RNAi worms were found to be significantly reduced in comparison to EV. In contrast, wah-1 RNAi worms had significantly extended lifespan. Moreover, RNAi knockdown of atfs-1 which leads to inactivation of UPRmt, did not change the lifespan of N2 worms with respect to EV. Upon blocking UPRmt by atfs-1 RNAi, the lifespan reduction conferred by pdhb-1 RNAi was significantly rescued towards normal lifespan. Metformin, however, significantly decreased the lifespan of all RNAis except pdhb-1, where it had no significant change in lifespan. Metformin also significantly suppressed the UPRmt at different adulthood days in ech-6 and wah-1 RNAi. Conclusion: Metformin extended the lifespan in C. elegans. The variable nature of lifespan in UPRmt invoked worms (ech-6, wah-1 and pdhb-1) delineates the complex nature of UPRmt in relation with the lifespan. Metformin suppressed UPRmt in ech-6 and wah-1 despite decreasing their lifespan. This lifespan reducing effect reiterated with atfs-1 and therefore hints that wildtype expression levels of ech-6, wah-1 and atfs-1 is required for metformin induced lifespan extension. Also, the amelioration of mitochondrial stress by the drug at specific time points during life might not be sufficient to confer longevity. The lifespan phenotype conferred by UPRmt in pdhb-1 was abolished when fed together with atfs-1 RNAi. This indicates the clear role of UPRmt in lifespan regulation. Interestingly, metformin did not change the lifespan of pdhb-1, which suggests that the drug might exert its effect by downregulating the function of pdhb-1.