A novel D2O tracer method to quantify RNA turnover as a biomarker of de novo ribosomal biogenesis, in vitro, in animal models, and in human skeletal muscle

Brook, Matthew S.; Wilkinson, D.J.; Mitchell, W. Kyle; Lund, Jonathan N.; Phillips, Bethan E.; Szewczyk, Nathaniel J.; Kainulainen, H.; Lensu, S.; Koch, L.G.; Britton, S.L.; Greenhaff, Paul L.; Smith, K.; Atherton, Philip J.

Abstract

Current methods to quantify in vivo RNA dynamics are limited. Here, we developed a novel stable isotope (D2O) methodology to quantify RNA synthesis (i.e., ribosomal biogenesis) in cells, animal models, and humans. First, proliferating C2C12 cells were incubated in D2O-enriched media and myotubes ±50 ng/ml IGF-I. Second, rat quadriceps (untrained, n = 9; 7-wk interval-“like” training, n = 13) were collected after ~3-wk D2O (70 atom %) administration, with body-water enrichment monitored via blood sampling. Finally, 10 (23 ± 1 yr) men consumed 150-ml D2O followed by 50 ml/wk and undertook 6-wk resistance exercise (6 × 8 repetitions, 75% 1-repetition maximum 3/wk) with body-water enrichment monitored by saliva sampling and muscle biopsies (for determination of RNA synthesis) at 0, 3, and 6 wk. Ribose mole percent excess (r-MPE) from purine nucleotides was analyzed via GC-MS/MS. Proliferating C2C12 cell r-MPE exhibited a rise to plateau, whereas IGF-I increased myotube RNA from 76 ± 3 to 123 ± 3 ng/μl and r-MPE by 0.39 ± 0.1% (both P < 0.01). After 3 wk, rat quadriceps r-MPE had increased to 0.25 ± 0.01% (P < 0.01) and was greater with running exercise (0.36 ± 0.02%; P < 0.01). Human muscle r-MPE increased to 0.06 ± 0.01 and 0.13 ± 0.02% at 3/6 wk, respectively, equating to synthesis rates of ~0.8%/day, increasing with resistance exercise to 1.7 ± 0.3%/day (P < 0.01) and 1.2 ± 0.1%/day (P < 0.05) at 3/6 wk, respectively. Therefore, we have developed and physiologically validated a novel technique to explore ribosomal biogenesis in a multimodal fashion.

Citation

Brook, M. S., Wilkinson, D., Mitchell, W. K., Lund, J. N., Phillips, B. E., Szewczyk, N. J., …Atherton, P. J. (2017). A novel D2O tracer method to quantify RNA turnover as a biomarker of de novo ribosomal biogenesis, in vitro, in animal models, and in human skeletal muscle. AJP - Endocrinology and Metabolism, 313(6), Article E681-E689. https://doi.org/10.1152/ajpendo.00157.2017

• Journal Article Type: Article
• Acceptance Date: Aug 14, 2017
• Online Publication Date: Aug 15, 2017
• Publication Date: Dec 1, 2017
• Deposit Date: Dec 6, 2017
• Publicly Available Date: Aug 16, 2018
• Journal: AJP: Endocrinology and Metabolism
• Print ISSN: 0193-1849
• Electronic ISSN: 1522-1555
• Publisher: American Physiological Society
• Peer Reviewed: Peer Reviewed
• Volume: 313
• Issue: 6
• Article Number: E681-E689
• DOI: https://doi.org/10.1152/ajpendo.00157.2017
• Keywords: Ribosomal biogenesis; D2O; RNA synthesis; Muscle
• Public URL: https://nottingham-repository.worktribe.com/output/897797
• Publisher URL: https://doi.org/10.1152/ajpendo.00157.2017