Healthspan‐Extending Treatments Enhance Proteostasis through Reduced Cellular Proliferation

Abstract

Aging is characterized by an accumulation of damaged intracellular components over time. Mechanisms that can prevent or delay the accumulation of intracellular damage may increase healthspan (i.e., the period of life spent free from chronic disease). Multiple long‐lived models demonstrate an increased ability to maintain protein homeostasis (proteostasis) over the duration of the lifespan. Rapamycin (Rap), and Metformin in combination with Rap (MetRap), have both been demonstrated to increase median lifespan. It is currently unknown how Rap and MetRap influence cellular proteostasis. Thus, the purpose of this study was to examine the influence of the healthspan‐extending treatments Rap and MetRap on protein turnover and cellular proliferation in vitro. We also sought to determine how autophagic flux contributes to protein turnover during Rap and MetRap treatments. We hypothesized that both Rap and MetRap would increase protein turnover (both synthesis and breadown) and reduce cellular proliferation. We further hypothesized that a decrease in autophagic flux would decrease protein turnover during Rap and MetRap treatments. Protein and DNA synthesis or breakdown rates were measured in C2C12 myotubes for up to 24h using deuterium oxide. Rapamycin (Rap 5 nM) and Rap in combination with Metformin (MetRap 2 mM/5 nM) were applied to C2C12 myotubes. Bafilomycin A1 (Baf 100 nM) was used to pre‐ and co‐treat myotubes to block autophagic flux during treatments. Fractional synthesis and breakdown rates were calculated for mitochondrial‐enriched, cytoplasmic‐enriched, and DNA fractions from each 100 mm culture dish in triplicate. Protein/DNA ratios (Pro/DNA) were calculated by dividing the fractional synthesis rate of proteins by DNA fractional synthesis rate to differentiate protein synthesis for growth versus maintaining proteostasis. In accordance with our hypotheses, we found that 24h of Rap treatment significantly increased mitochondrial (P<0.01) and cytoplasmic (P<0.01) Pro/DNA compared to Con. Inhibition of autophagic flux reduced mitochondrial protein breakdown rate by 29% compared to CON (P<0.001), while cytoplasmic protein breakdown was only reduced by 9% compared to CON (P<0.05). Mitochondrial Pro/DNA during Rap and MetRap was not altered by co‐treatment with Baf. However, Baf+Rap significantly increased (P<0.0001) cytoplasmic Pro/DNA compared to Rap as well as compared to CON (P<0.0001). Further, Baf+MetRap significantly increased (P<0.0001) cytoplasmic Pro/DNA compared to MetRap. Baf+Rap and Baf+MetRap significantly reduced cellular DNA synthesis compared to CON, Rap, and MetRap (all P<0.001). Overall, these data suggest that Rap and MetRap improve mitochondrial and cytoplasmic proteostatic mechanisms in vitro, and surprisingly, inhibition of autophagy did not have a negative impact on cytoplasmic proteostatic mechanisms.Support or Funding InformationThis project is supported by NIH‐NIA‐AG‐042569 (BFM and KLH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.