Abstract: Manipulating Skeletal Muscle Acetylation to Enhance Healthspan and LifespanHuman aging is marked by a progressive loss of skeletal muscle mass and cognitive function—two primary determinants of healthspan and lifespan. Even though there are many studies characterizing this association, the mechanisms underlying this relationship remain unclear. In contrast to aging, endurance exercise has been shown to improve skeletal muscle mass and function and slow age-related cognitive decline 1,2. Further, animals bred for low levels of physical activity show impaired muscle and neurocognitive function 3. The main adaptations seen with endurance exercise training are heightened fat oxidation, and increased mitochondrial mass and activity downstream of a transcriptional co-factor called the peroxisome-proliferator activates receptor 1α (PGC-1α), which is regulated by acetylation 4,5. Roberts et al (2017) demonstrated that the use of a ketogenic diet (KD), increased protein acetylation and activated PGC-1α in muscle concomitant with improved markers of skeletal muscle strength, mitochondrial mass and activity, endurance, neurocognitive function, and extended lifespan 13.6% in mice 6. Along with enhanced healthspan and lifespan, mice on a KD demonstrated improved muscle mitochondrial biogenesis, muscle mass, and oxidative fiber cross-sectional area; all things associated with endurance exercise training 7. However, the molecular mechanisms underlying the benefits seen with increases in either exercise or a KD on skeletal muscle and neurocognitive function remains largely unexplored. Our global hypothesis for this body of work was that increasing protein acetylation plays a central role in improving skeletal muscle mass and function, mitochondrial biogenesis, and neurocognitive function. In the present work, we identify novel mechanisms that underlie the ability of skeletal muscle to increase muscle mass and function, and describe the role of skeletal muscle mitochondria in augmenting cognitive performance in response to manipulation of acetylation either through natural compounds, pharmaceuticals, or a KD. Our approach to understand how acetylation both with and without a KD enhances healthspan, and lifespan was 4-fold: 1) Determine the effect of a cocktail of natural products, selected to inhibit the NAD-dependent deacetylase Sirtuin 1 (SIRT1) in rats following ablation of the gastrocnemius and soleus muscles (Functional Overload—FO), on the resulting increase in plantaris muscle fiber cross sectional area, 2) Establish how the class II histone deacetylase (HDAC) inhibitor, scriptaid, affects skeletal muscle and neurocognitive function, and 3) Determine whether a 2-month KD alters skeletal muscle function, mitochondrial mass, and cognitive behavior in middle-aged female mice, Muscle mass and strength are important aspects of human health. Low levels of muscle mass and strength are directly correlated with lifespan. In Chapter 2, we optimized a cocktail of naturally occurring compounds (epicatechin, epicatechin-gallate, and celestrol) that were believed to inhibit sirtuin 1. This work shows that optimal inhibition of SIRT1 increases global protein acetylation (Figure 2.3C), and the increase in fiber cross sectional area (fCSA) in response to FO 61.5% more than vehicle dosed counterparts (Figure 2.2H). One proposed limit to skeletal muscle hypertrophy in both mice and man is the capacity for protein synthesis (i.e., ribosome mass)8–10. In response to an optimal dose of the SIRT1 inhibitor cocktail, ribosomal RNA (which comprises ~80% of the total RNA) relative to muscle mass decreased in the optimal group (Figure 2.4B). This was supported by lower markers of ribosome biogenesis (ITS1 and 5’ETS RNA levels) indicating that the benefit of the cocktail is not the result of increased translational machinery (Figure 2.4C&D). One possible explanation for the apparent increase in hypertrophy could be an increase in translational efficiency secondary to acetylation of ribosomes 11. In support of this, immunoprecipitation of ribosomal subunits showed greater associated acetylated lysine levels in the optimal group when compared to control animals (Figure 2.6A-C). Though a long term KD has been shown to help maintain oxidative fCSA and improve muscle and cognitive metrics, there is still uncertainty surrounding the mechanism underlying the changes caused by a KD 6,7. To address this gap in knowledge, in Chapter 3 we employed an isocaloric (11.2 kcal/day) KD in 23-month-old mice (Table 3.2) together with concomitant injection of either the class II HDAC inhibitor scriptaid (SCRIPT) or a vehicle (VEH) control. Our findings demonstrated that a one-month KD intervention increases mitochondrial mass (Figure 3.2D) and improved grip strength (Figure 3.1G) in skeletal muscle preferentially while not affecting fCSA (Figure 3.4A&B). As an explanation for the significant improvement on grip strength in the KD fed animals, we are the first to report that an acute KD increases dystrophin protein levels (Figure 3.4F). These data indicate that KD muscle is better able to laterally transmit force and prevent contraction induced muscle damage than their CD fed counterparts. To understand to what degree a short-term KD started late in life influences cognitive behavior the Y-Maze, Novel Object Recognition, and Open Field assays were conducted (Figure 3.1H, 3.1J&K). We report no changes between the CD and KD fed animals. These findings indicate that an acute KD first effects skeletal muscle biochemistry and functionality prior to that of neurocognitive function. Interestingly, all of the biochemical and functional benefits seen with an acute KD were blunted when paired with SCRIPT treatment. This blunting of adaptation coincides with KD SCRIPT animals’ inability to produce adequate ketones in the fasted state. This finding implies that ketone production may be a crucial component of maintaining skeletal muscle adaptation and functionality in aged female mice. Even though positive effects of a KD had been demonstrated in old and diseased male mice, whether the diet could benefit females was uncertain. To test the hypothesis that female mice would thrive on a KD, in Chapter 4 middle-aged female mice were put on a two-month isocaloric KD (Table 4.1) and upon collection, skeletal muscle and liver mitochondrial mass and biogenesis, cognitive function, and serum were analyzed. Our results show that a two-month KD is sufficient to improve mitochondrial mass (Figure 4.4A) and PGC-1 protein level in the total and nuclear muscle fractions (Figure 4.4C&E) of skeletal muscle. PGC-1α being responsible for the expression of kynurenine aminotransferases (KAT) in muscle 12, enzymes that convert a potential neurotoxin kynurenine (KYN) into kynurenic acid (KYNA) that cannot enter the brain, therefore we measured KAT protein levels in muscle after a 2-month KD. Two KAT proteins, KAT1 and KAT4 increased in the GTN muscle (Figure 4G&I). The increase in muscle KAT proteins was associated with a significant decrease in circulating KYN and strong trend for KYNA to increase (Figure 4F&G). The increase in KAT protein and decrease in circulating KYN were associated with improvements in Barnes maze and rearing assays, indicating greater spatial learning and memory capacity along with decreased anxiousness (Figure 4J&K). We are the first to propose a mechanism that connects muscle metabolism to brain function in response to a dietary intervention. Within this dissertation, I discuss mechanisms whereby altering acetylation in skeletal muscle improves translational efficiency resulting in greater load-induced skeletal muscle hypertrophy; however, acetylation is not sufficient to induce mitochondrial biogenesis or improve cognitive function.