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
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...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
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American Physiological Society
2017
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| Online Access: | https://eprints.nottingham.ac.uk/48573/ |
| _version_ | 1848797797181030400 |
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| author | 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. |
| author_facet | 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. |
| author_sort | Brook, Matthew S. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T20:09:35Z |
| format | Article |
| id | nottingham-48573 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T20:09:35Z |
| publishDate | 2017 |
| publisher | American Physiological Society |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-485732020-05-04T19:20:20Z https://eprints.nottingham.ac.uk/48573/ 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. 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. American Physiological Society 2017-12-01 Article PeerReviewed 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. 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 Ribosomal biogenesis; D2O; RNA synthesis; Muscle https://doi.org/10.1152/ajpendo.00157.2017 doi:10.1152/ajpendo.00157.2017 doi:10.1152/ajpendo.00157.2017 |
| spellingShingle | Ribosomal biogenesis; D2O; RNA synthesis; 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. 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 |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Ribosomal biogenesis; D2O; RNA synthesis; Muscle |
| url | https://eprints.nottingham.ac.uk/48573/ https://eprints.nottingham.ac.uk/48573/ https://eprints.nottingham.ac.uk/48573/ |