Utilizing Nonequilibrium Isotope Enrichments to Dramatically Increase Turnover Measurement Ranges in Single Biopsy Samples from Humans.

The synthesis of new proteins and the degradation of old proteins in vivo can be quantified in serial samples using metabolic isotope labeling to measure turnover. Because serial biopsies in humans are impractical, we set out to develop a method to calculate the turnover rates of proteins from single human biopsies. This method involved a new metabolic labeling approach and adjustments to the calculations used in previous work to calculate protein turnover. We demonstrate that using a nonequilibrium isotope enrichment strategy avoids the time dependent bias caused by variable lag in label delivery to different tissues observed in traditional metabolic labeling methods. Turnover rates are consistent for the same subject in biopsies from different labeling periods, and turnover rates calculated in this study are consistent with previously reported values. We also demonstrate that by measuring protein turnover we can determine where proteins are synthesized. In human subjects a significant difference in turnover rates differentiated proteins synthesized in the salivary glands versus those imported from the serum. We also provide a data analysis tool, DeuteRater-H, to calculate protein turnover using this nonequilibrium metabolic 2H2O method.

Sex differences in changes of protein synthesis with rapamycin treatment are minimized when metformin is added to rapamycin.

Loss of protein homeostasis is a hallmark of the aging process. We and others have previously shown that maintenance of proteostasis is a shared characteristic of slowed-aging models. Rapamycin (Rap) exerts sex-specific effects on murine lifespan, but the combination of Rap with the anti-hyperglycemic drug metformin (Rap + Met) equally increases male and female mouse median lifespan. In the current investigation, we compare the effects of short-term (8 weeks) Rap and Rap + Met treatments on bulk and individual protein synthesis in two key metabolic organs (the liver and skeletal muscle) of young genetically heterogeneous mice using deuterium oxide. We report for the first time distinct effects of Rap and Rap + Met treatments on bulk and individual protein synthesis in young mice. Although there were decreases in protein synthesis as assessed by bulk measurements, individual protein synthesis analyses demonstrate there were nearly as many proteins that increased synthesis as decreased synthesis rates. While we observed the established sex- and tissue-specific effects of Rap on protein synthesis, adding Met yielded more uniform effects between tissue and sex. These data offer mechanistic insight as to how Rap + Met may extend lifespan in both sexes while Rap does not.