Hamilton, Karyn L.

Abstract17α-Estradiol (17α-E2) is a “non-feminizing” estrogen that extends life span in male, but not female, mice. We recently reported that 17α-E2 had robust beneficial effects on metabolic and inflammatory parameters in aged male mice. However, it remains unclear if 17α-E2 also delays other “hallmarks” of aging, particularly maintaining proteostasis. Here, we used isotope labeling methods in older mice to examine proteostatic mechanisms. We compared weight-matched mild calorie restricted (CR) and 17α-E2 treated male mice with the hypothesis that 17α-E2 would increase protein synthesis for somatic maintenance. 17α-E2 had no effect on protein synthesis or DNA synthesis in multiple tissues, including white adipose tissue. Conversely, mild short-term CR decreased DNA synthesis and increased the protein to DNA synthesis ratio in multiple tissues. Examination of individual protein synthesis and content did not differentiate treatments, although it provided insight into the regulation of protein content between tissues. Contrary to our hypothesis, we did not see the predicted differences in protein to DNA synthesis following 17α-E2 treatment. However, mild short-term CR elicited differences consistent with both lifelong CR and other treatments that curtail aging processes. These data indicated that despite similar maintenance of body mass, 17α-E2 and CR treatments elicit distinctly different proteostatic outcomes.

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10–12. High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H2O2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance.