Accounting for non‐steady state conditions during long‐term deuterium oxide‐based measurement of protein turnover
Authors: Musci Robert V. , Konopka Adam R. , Wolff Christopher A. , Reid Justin J. , Laurin Jaime L. , Peelor Frederick F. , Shipman Patrick D. , Hamilton Karyn L. , Miller Benjamin F. 
Published: June 2021

Introduction

Most isotope calculations to measure synthesis rates require steady state conditions. In skeletal muscle, steady state refers to a period of time in which there is neither loss or gain of protein. Deuterium oxide (D2O) is a stable isotopic tracer that can measure protein synthesis over long periods of time. This is advantageous when measuring changes in protein synthesis in response to long‐term interventions such as exercise training or pharmacologic interventions. However, prolonging the period of measurement increases the likelihood of non‐steady state conditions. The purpose of this study was to demonstrate that non‐steady state conditions such as growth can change interpretation of tracer data and validate a model that accounts for non‐steady state conditions when assessing muscle protein synthesis. We hypothesized that use of the appropriate model can account for non‐steady state conditions when measuring muscle protein synthesis.

Methods

To demonstrate the potential implications of non‐steady state conditions, we used D2O to measure skeletal muscle protein synthesis over two weeks in three month old male Sprague Dawley rats with access to running wheels during a period of growth. Then we established a model to account for non‐steady state conditions and validated the model in cultured C2C12 myoblasts and myotubes during periods of cell growth and atrophy. Finally, we applied this model in vivo by measuring protein synthesis in four month old male CB6F1 mice with steady weight or weight loss over a month.

Results

During a period of growth, active rats gained less body mass than their sedentary counterparts. There was a significant (p<0.05) positive relation between changes in body size and fractional synthesis rates (FSR) (R2 = 0.50, 0.475, 0.50 for myofibrillar, cytosolic, and mitochondrial FSR, respectively) that obscured any exercise‐related increases in protein synthesis. In vitro studies validated the model parameters that rely on a change in protein mass. We grouped mice based on weight maintenance versus weight loss during the period of measurement. We used our modeling parameters to estimate changes in protein synthesis during the steady and non‐steady conditions.

Conclusions

Non‐steady state conditions occur with greater likelihood in long‐ versus short‐term protein synthesis studies and should be accounted for. We showed that a period of growth could account for ~50% of all variation in changes in protein synthesis in all subfractions of skeletal muscle, and this growth effect could mask more subtle effects that may be important for hypothesis testing. We have developed a model to account for non‐steady state conditions over long‐term interventions. We applied this model to a one month intervention to account for body mass changes in response to dietary restriction. By applying this model to future long‐term interventions, we can more accurately discern the effects of interventions on protein synthesis in non steady‐state conditions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.