Description: Temperature dependent growth is an important indicator to understand the thermal tolerance of organisms and to project their vulnerability to future climate change. Direct measurements of temperature dependent weight gains, however, are experimentally challenging and time consuming in long-lived species. Here, we reassess methodology to quantify the in vivo protein synthesis rate from amino acids, as a key component of growth. We developed an analytical method that is both robust against analytical errors and does not require hazardous radioactive materials. We utilized the incorporation of isotopically 13C15N-labeled-phenylalanine into fish muscle followed by quantification by liquid chromatography mass spectrometry to calculate accurate net protein synthesis rates in muscle tissue of Antarctic fish, Pachycara brachycephalum, in vivo. Specifically, we injected 150 mM of 13C9H915N1 phenylalanine intraperitoneally and sampled muscle tissue in 1,5h steps between 0 and 6 hours after injection. We quantified labeled and unlabeled phenylalanine both in muscle protein and in the cytosol. This allowed us to critically re-evaluate three different protein synthesis rate (Ks) calculation methodologies that have been developed over the last decades. The calculated values differ by more than 70-fold (0.048 ± 0.021% day-1 up to 3.56 ± 2.16 day-1) between methods. We argue that the Ks calculation including a proportionate ratio of protein synthesis from (unlabeled) free amino acids yields the most realistic Ks values for cold water fish. Eventually, the standardization of the net protein synthesis rate calculation will lead to dependable quantitative representations of organismal stress in response to climate change.

Description: Protein turnover is highly energy consuming and overall relates to an organism's growth performance varying largely between species, e.g., due to pre-adaptation to environmental characteristics such as temperature. Here, we determined protein synthesis rates and capacity of protein degradation in white muscle of the cold stenothermal Antarctic eelpout (Pachycara brachycephalum) and its closely related temperate counterpart, the eurythermal common eelpout (Zoarces viviparus). Both species were exposed to acute warming (P. brachycephalum, 0 °C + 2 °C/day; Z. viviparus, 4 °C + 3 °C/day). The in vivo protein synthesis rate (Ks) was monitored after injection of 13C-phenylalanine, and protein degradation capacity was quantified by measuring the activity of cathepsin D in vitro. Untargeted metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy was used to identify the metabolic processes involved. Independent of temperature, the protein synthesis rate was higher in P. brachycephalum (Ks = 0.38–0.614 %/day) than in Z. viviparus (Ks= 0.148-0.379%/day). Whereas protein synthesis remained unaffected by temperature in the Antarctic species, protein synthesis in Z. viviparus increased to near the thermal optimum (16 °C) and tended to fall at higher temperatures. Most strikingly, capacities for protein degradation were about ten times higher in the Antarctic compared to the temperate species. These differences are mirrored in the metabolic profiles, with significantly higher levels of complex and essential amino acids in the free cytosolic pool of the Antarctic congener. Together, the results clearly indicate a highly cold-compensated protein turnover in the Antarctic eelpout compared to its temperate confamilial. Constant versus variable environments are mirrored in rigid versus plastic functional responses of the protein synthesis machinery.