Exercise mimicry: Characterization of nutraceutical agents that may contribute to mitochondrial homeostasis in skeletal muscle

Abstract

While it is well established that endurance exercise improves mitochondrial health in skeletal muscle, optimizing nutraceutical agents to mimic these metabolic achievements in the cell remains a challenge. Nonetheless, the application of exercise mimetics is a fruitful direction to pursue, as they may target and activate the same mechanisms that are upregulated with exercise administration alone. This is particularly useful under conditions where contractile activity is compromised due to muscle disuse, disease, or aging. The agents Sulforaphane (SFN) and Urolithin A (UroA) represent our preliminary candidates for antioxidation and mitophagy, respectively, for maintaining mitochondrial turnover and homeostasis. SFN is a powerful inducer of the Nrf‐2‐ARE pathway, a mechanism that upregulates cellular defences against oxidative stress. On the other hand, the metabolite, UroA, has developed a reputable role in regulating mitochondrial turnover via mitophagy. The purpose of this ongoing study is to characterize these nutraceutical agents both in their time‐ and dose‐dependent capacities to induce changes in protein content relative to the antioxidant and mitophagy pathways, in C2C12 myotubes. Differentiated muscle cells were treated with two concentrations of each nutraceutical for 4 h, 24 h, or 48 h. Immunoblot analysis was conducted to measure changes in protein content. SFN treatment after 4 h rendered a marked increase in the master regulator for antioxidation, and transcription factor, Nrf‐2, with no apparent changes in its negative regulator Keap‐1 at any given time point. Nuclear‐cytoplasmic fractions confirmed Nrf‐2 translocation to the nucleus following 4 h of treatment, likely representing the potent activation of antioxidant genes. This aligns with the upregulation of the downstream antioxidant markers HO‐1 and NQO1, showing 4‐5‐fold increases as early as 4 h and 24 h, respectively, and maintained after 48 h. Despite modest effects with Urolithin A, some significant changes took place under basal conditions. The upstream kinase phospho‐AMPK was activated by 2‐fold as early as 4 hr of treatment with UroA, which subsided by 24 and 48 h. Previous reports observed changes with the agent on autophagy markers, which includes the modest 1.3‐fold increases in p62 and LC3‐II after 48 h of treatment. However, no observable changes took place with respect to the mitophagy markers phospho‐ULK1, Parkin, or PINK1. Nonetheless, our preliminary results suggest that these agents may be suitable candidates as exercise mimetics. These data also set the stage for an examination of the synergistic effect of these nutraceuticals, in combination with contractile activity, on mitochondrial turnover and function.