Joshua C. Drake

Assessment of Mitochondrial Biogenesis and mTORC1 Signaling During Chronic Rapamycin Feeding in Male and Female Mice

Chronic inhibition of the protein synthesis regulator mTORC1 through rapamycin extends life span in mice, with longer extension in females than in males. Whether rapamycin treatment inhibits protein synthesis or whether it does so differently between sexes has not been examined. UM-HET3 mice were fed a control or rapamycin-supplemented (Rap) diet for 12 weeks. Protein synthesis in mixed, cytosolic (cyto), and mitochondrial (mito) fractions and DNA synthesis and mTORC1 signaling were determined in skeletal muscle, heart, and liver. In both sexes, mito protein synthesis was maintained in skeletal muscle from Rap despite decreases in mixed and cyto fractions, DNA synthesis, and rpS6 phosphorylation. In the heart, no change in protein synthesis occurred despite the decreased DNA synthesis. In the heart and liver, Rap males were more sensitive to mTORC1 inhibition than Rap females. In conclusion, we show changes in protein synthesis and mTORC1 signaling that differ by sex and tissue.

Calorie Restriction Does Not Increase Short-term or Long-term Protein Synthesis

Increased protein synthesis is proposed as a mechanism of life-span extension during caloric restriction (CR). We hypothesized that CR does not increase protein synthesis in all tissues and protein fractions and that any increased protein synthesis with CR would be due to an increased anabolic effect of feeding. We used short- (4 hours) and long-term (6 weeks) methods to measure in vivo protein synthesis in lifelong ad libitum (AL) and CR mice. We did not detect an acute effect of feeding on protein synthesis while liver mitochondrial protein synthesis was lower in CR mice versus AL mice. Mammalian target of rapamycin (mTOR) signaling was repressed in liver and heart from CR mice indicative of energetic stress and suppression of growth. Our main findings were that CR did not increase rates of mixed protein synthesis over the long term or in response to acute feeding, and protein synthesis was maintained despite decreased mTOR signaling.

Nrf2 Signaling and the Slowed Aging Phenotype: Evidence from Long-Lived Models

Studying long-lived animals provides novel insight into shared characteristics of aging and represents a unique model to elucidate approaches to prevent chronic disease. Oxidant stress underlies many chronic diseases and resistance to stress is a potential mechanism governing slowed aging. The transcription factor nuclear factor (erythroid-derived 2)-like 2 is the “master regulator” of cellular antioxidant defenses. Nrf2 is upregulated by some longevity promoting interventions and may play a role in regulating species longevity. However, Nrf2 expression and activity in long-lived models have not been well described. Here, we review evidence for altered Nrf2 signaling in a variety of slowed aging models that accomplish lifespan extension via pharmacological, nutritional, evolutionary, genetic, and presumably epigenetic means.

Long-lived crowded-litter mice have an age-dependent increase in protein synthesis to DNA synthesis ratio and mTORC1 substrate phosphorylation

Increasing mouse litter size [crowded litter (CL)] presumably imposes a transient nutrient stress during suckling and extends lifespan through unknown mechanisms. Chronic calorically restricted and rapamycin-treated mice have decreased DNA synthesis and mTOR complex 1 (mTORC1) signaling but maintained protein synthesis, suggesting maintenance of existing cellular structures. We hypothesized that CL would exhibit similar synthetic and signaling responses to other long-lived models and, by comparing synthesis of new protein to new DNA, that insight may be gained into the potential preservation of existing cellular structures in the CL model. Protein and DNA synthesis was assessed in gastroc complex, heart, and liver of 4- and 7-mo CL mice. We also examined mTORC1 signaling in 3- and 7-mo aged animals. Compared with controls, 4-mo CL had greater DNA synthesis in gastroc complex with no differences in protein synthesis or mTORC1 substrate phosphorylation across tissues. Seven-month CL had less DNA synthesis than controls in heart and greater protein synthesis and mTORC1 substrate phosphorylation across tissues. The increased new protein-to-new DNA synthesis ratio suggests that new proteins are synthesized more so in existing cells at 7 mo, differing from 4 mo, in CL vs. controls. We propose that, in CL, protein synthesis shifts from being directed toward new cells (4 mo) to maintenance of existing cellular structures (7 mo), independently of decreased mTORC1.

Long-lived Snell dwarf mice display increased proteostatic mechanisms that are not dependent on decreased mTORC1 activity

Maintaining proteostasis is thought to be a key factor in slowed aging. In several growth‐restricted models of long‐life, we have shown evidence of increased proteostatic mechanisms, suggesting that proteostasis may be a shared characteristic of slowed aging. The Snell dwarf mouse is generated through the mutation of the Pit‐1 locus causing reductions in multiple hormonal growth factors and mTORC1 signaling. Snell dwarfs are one of the longest lived rodent models of slowed aging. We hypothesized that proteostatic mechanisms would be increased in Snell compared to control (Con) as in other models of slowed aging. Using D2O, we simultaneously assessed protein synthesis in multiple subcellular fractions along with DNA synthesis in skeletal muscle, heart, and liver over 2 weeks in both sexes. We also assessed mTORC1‐substrate phosphorylation. Skeletal muscle protein synthesis was decreased in all protein fractions of Snell compared to Con, varied by fraction in heart, and was not different between groups in liver. DNA synthesis was lower in Snell skeletal muscle and heart but not in liver when compared to Con. The new protein to new DNA synthesis ratio was increased threefold in Snell skeletal muscle and heart compared to Con. Snell mTORC1‐substrate phosphorylation was decreased only in heart and liver. No effect of sex was seen in this study. Together with our previous investigations in long‐lived models, we provide evidence further supporting proteostasis as a shared characteristic of slowed aging and show that increased proteostatic mechanisms may not necessarily require a decrease in mTORC1.

Participation in a 1,000-mile race increases the oxidation of carbohydrate in Alaskan sled dogs

The Alaskan Husky has been specifically bred for endurance performance and is capable of extreme endurance performance. We examined sled dogs in the trained state at the beginning of the race season and after a 1,600-km race (Iditarod). Our hypothesis was that lipids would be the predominant substrate during submaximal exercise in long-distance racing sled dogs, and a 1,600-km race would increase the reliance on lipids during an exercise bout at the same absolute exercise intensity. The experiments were completed over three testing periods, which were completed in January of two different years before participation in a 1,600-km race, or in March shortly after completion of a 1,600-km race. After determination of H(13)CO3 (-) recovery, the dogs were tested with primed continuous infusions of [1,1,2,3,3-(2)H]glycerol, [3-(13)C]lactate, or [6,6-(2)H2]glucose. During exercise, respiratory exchange ratio was significantly higher in raced (0.92 ± 0.01) compared with nonraced (0.87 ± 0.01) dogs. During exercise, glucose rate of appearance was potentially sustained by a large glycerol rate of disappearance with an increase in lactate rates of oxidation after a 1,600-km race. Therefore, contrary to our hypothesis, the sled dogs were dependent on carbohydrate energy sources, a reliance that increased further after participation in a 1,600-km race.

Assessment of protein synthesis in highly aerobic canine species at the onset and during exercise training

Canis lupus familiaris, the domesticated dog, is capable of extreme endurance performance. The ability to perform sustained aerobic exercise is dependent on a well-developed mitochondrial reticulum. In this study we examined the cumulative muscle protein and DNA synthesis in groups of athletic dogs at the onset of an exercise training program and following a strenuous exercise training program. We hypothesized that both at the onset and during an exercise training program there would be greater mitochondrial protein synthesis rates compared with sedentary control with no difference in mixed or cytoplasmic protein synthesis rates. Protein synthetic rates of three protein fractions and DNA synthesis were determined over 1 wk using (2)H2O in competitive Alaskan Huskies and Labrador Retrievers trained for explosive device detection. Both groups of dogs had very high rates of skeletal muscle protein synthesis in the sedentary state [Alaskan Huskies: Mixed = 2.28 ± 0.12, cytoplasmic (Cyto) = 2.91 ± 0.10, and mitochondrial (Mito) = 2.62 ± 0.07; Labrador Retrievers: Mixed = 3.88 ± 0.37, Cyto = 3.85 ± 0.06, and Mito = 2.92 ± 0.20%/day]. Mitochondrial (Mito) protein synthesis rates did not increase at the onset of an exercise training program. Exercise-trained dogs maintained Mito protein synthesis during exercise training when mixed (Mixed) and cytosolic (Cyto) fractions decreased, and this coincided with a decrease in p-RpS6 but also a decrease in p-ACC signaling. Contrary to our hypothesis, canines did not have large increases in mitochondrial protein synthesis at the onset or during an exercise training program. However, dogs have a high rate of protein synthesis compared with humans that perhaps does not necessitate an extra increase in protein synthesis at the onset of aerobic exercise training.