Metformin-Regulated Mitochondrial Peptides and their Effects on Aging

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Metformin has healthspan and lifespan enhancing effects in model organisms and is a candidate therapy for aging in humans. The exact mechanism of action of metformin is poorly understood, but the mitochondrion is clearly a critical metformin target involving modulation of respiratory-chain function attributed to inhibition of Complex-1 as well as other mitochondrial actions on fission, reactive oxygen species production, etc. Our lab described the presence of multiple small open reading frames (smORFs) within mitochondrial DNA that encode a novel class of mitochondrial-derived peptides (MDPs). These MDPs have diverse cellular actions, often leading to improved metabolism, cytoprotection, and healthspan. We also demonstrated that mitochondrial single nucleotide polymorphisms (mtSNPs) within these MDPs change their activity and increase risk of aging-related diseases. Recently, we developed a novel bioinformatic approach to assess changes in the expression of the entire mitochondrial smORFome called MDPseq. Using MDPSeq, we can demonstrate profound changes in the expression of MDPs in disease states as well as during aging. We re-analyzed multiple publicly available RNAseq datasets of humans and mice treated with metformin and observed dramatic changes in the MDP smORF RNA transcript levels. After synthesizing the MDPs most potently regulated by metformin and comparing them to metformin in several in vitro systems, we identified a number of candidate metformin-induced MDPs that act in a metformin-mimetic fashion. One of these MDPs, named Metformin-stimulated Mitochondrial-Derived Peptide (Ms.MDP), has similar activity to metformin on glucose metabolism, mitochondrial function, and AMPK signaling. Ms.MDP siRNA diminishes some of metformin actions. When administered to mice fed a high-fat diet, Ms.MDP significantly attenuated weight-gain and dramatically reduced blood sugar, ALT, AST, and multiple inflammatory cytokines similarly – or more potently – than metformin. Thus, we hypothesize that a key mechanism of metformin is MDP regulation, and that these metformin-regulated MDPs mediate some of the functions of metformin. We further hypothesize that MDPs are crucially involved in the anti-aging effects of metformin, which in turn can potentially help define individuals that will respond to metformin and possibly serve as alternative healthspan-enhancing treatments. Our specific aims to test this are as follows: 1: Identify human MDP smORF RNA transcripts in liver that are regulated by metformin, and mitochondrial DNA variants that determine the clinical response to metformin in several large human cohorts. 2: Compare the effects of aging and metformin on the MDP expression signature in mice. 3: Characterize the aging-related effects of metformin-regulated MDPs in vitro, in various cell types, and 4: Examine the in vivo mechanisms and therapeutic potential of chronic administration of promising metformin- regulated MDPs in aged mice. Together, these studies will define a completely novel direction in understanding the actions of metformin on delaying aging, and will establish novel biomarkers as well as complementary/alternative anti-aging approaches.