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, M.S. and Wilkinson, D.J. and Mitchell, W. Kyle and Lund, Jonathan N. and Phillips, Bethan E. and Szewczyk, Nathaniel J. and Kainulainen, H. and Lensu, S. and Koch, L.G. and Britton, S.L. and Greenhaff, Paul L. and Smith, K. and Atherton, Philip 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). E681-E689. ISSN 1522-1555

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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.

Item Type: Article
Keywords: Ribosomal biogenesis; D2O; RNA synthesis; Muscle
Schools/Departments: University of Nottingham, UK > Faculty of Medicine and Health Sciences > School of Medicine > Division of Medical Sciences and Graduate Entry Medicine
University of Nottingham, UK > Faculty of Medicine and Health Sciences > School of Life Sciences
Identification Number: 10.1152/ajpendo.00157.2017
Depositing User: Eprints, Support
Date Deposited: 06 Dec 2017 11:09
Last Modified: 08 Dec 2017 10:57
URI: http://eprints.nottingham.ac.uk/id/eprint/48573

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