Determining the influence of metformin treatment on mitochondrial calcium flux during aerobic exercise training of healthy, aging rats

Project Summary Metformin (MET), the most widely prescribed medication for treating type 2 diabetes, is increasingly recognized for its healthspan extension effects. If MET is to be an effective healthspan extending drug, it is critical to understand the effects of MET during periods of relative health and how it interacts with other healthspan-related interventions such as exercise. Studies that have examined the MET-exercise interaction overwhelmingly show that MET inhibits the positive effects of aerobic exercise training (AET), but the mechanisms behind this effect are still unknown. Our recent clinical trial also demonstrated that subjects with the highest mitochondrial complex 1 supported respiratory function and insulin sensitivity were the most negatively affected by MET treatment with an exercise intervention. The proposed study will determine if the detrimental effects of MET treatment on skeletal muscle adaptations to AET involves altered calcium handling. Calcium handling is imperative for mitochondrial function, adaptations to exercise, and muscle health during aging. This project is significant because it further defines the context-specificity of MET benefits on healthspan. We hypothesize that MET treatment will impede calcium flux into the mitochondria during exercise training in aging rats, inhibiting the positive adaptations to AET. To test this hypothesis, we will use a rat model with genetic selection for high intrinsic aerobic capacity (HCR) because they represent, by many outcomes, healthy human subjects. The Specific Aims are to: 1) Determine how MET treatment alters mitochondrial calcium flux during AET and 2) Determine if MET treatment during AET alters the turnover of calcium handling proteins and structures regulating calcium kinetics. We will assess in vivo cytosolic and mitochondrial calcium kinetics using 2-photon fluorescence microscopy. Changes to mitochondrial calcium flux and function of skeletal muscle with and without 12 weeks of AET and/or MET will be measured using an Oroboros O2K. To determine if MET changes adaptations to AET, we will develop a targeted mitochondrial proteomics panel to measure the quantity and turnover of mitochondrial and calcium-handling proteins. We will also use a novel optic clearing technique in combination with light sheet microscopy to establish changes to mitochondrial-associated membrane contacts (MAM) structure and location with and without MET. We expect that with MET treatment, animals will have a lower rate of mitochondrial calcium uptake coupled with lower respiration, and that this inhibition will be more prominent in the exercised animals. Additionally, we expect MET will negatively impact the mitochondrial proteome related to calcium flux and hinder improvements associated with MAM number and distance to calcium release units in response to exercise. Successful completion of these aims will allow us to uncover mechanisms that contribute to the inhibition of AET adaptations with MET treatment in aging animals. Key training aspects include novel bioenergetic, flux and proteomic assessments of calcium handling in mitochondria that are crucial for my development to an independent investigator focused on targeting mitochondria for muscle aging.