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

Abstract

50ml/wk and undertook 6-wks resistance-exercise (RE; 6×8 repetitions, 75%-1RM 37 3/wk) with BWE monitored by saliva sampling and muscle biopsies (for determination 38 of RNA synthesis) 0-3-6-wks. Ribose mole percent excess (r-MPE) from purine 39 nucleotides was analyzed via GC-MS/MS. Proliferating C2C12 cells r-MPE exhibited a 40 rise-to-plateau while IGF-1 increased myotube RNA from 76±3ng/ul to 123±3ng/ul and 41 r-MPE by 0.39±0.1% (both P<0.01). After 3-wks, rat quadriceps r-MPE had increased 42 to 0.25±0.01% (P<0.01) and was greater with running-exercise (0.36±0.02%; P<0.01)). 43Human muscle r-MPE increased to 0.06±0.01% and 0.13±0.02% at 3/6-wks 44 respectively equating to synthesis rates of ~0.8%/d, increasing with RE to 1.7±0.3%/d 45 (P<0.01) and 1.2±0.1%/d (P<0.05) at 3/6-wks, respectively. Therefore, we have 46 developed and physiologically validated a novel technique to explore ribosomal 47 biogenesis in a multi-modal fashion.

Physiological Differences Between Low Versus High Skeletal Muscle Hypertrophic Responders to Resistance Exercise Training: Current Perspectives and Future Research Directions

“…In rats, recent evidence suggests the degree of synergist ablation-induced plantaris hypertrophy parallels proportional increases in ribosome content ( Nakada et al, 2016 ). Our laboratory ( Mobley et al, 2016 , 2018b ; Roberts et al, 2016 ) and others ( West et al, 2016 ; Brook et al, 2017 ) have also reported that acute and chronic resistance exercise training models in rats increase indices of ribosome biogenesis and ribosome content, respectively.…”

“…Conversely, low responders experienced a small but significant increase in VL muscle thickness (+4%) coupled with an 8% non-significant increase in ribosome content following training ( p = 0.25). Interestingly, Brook et al (2017) recently employed the D 2 O tracer method to examine the rate of ribosome biogenesis during 6 weeks of resistance exercise training in college-aged males. These authors reported: (a) basal ribosome synthesis rates were ∼0.8% per day, whereas synthesis rates increased during the training period to ∼1.7% per day, and (b) increased basal ribosome biogenesis rates during training were significantly correlated to increased basal MPS rates ( r = 0.57, p < 0.01).…”

The age-related loss of skeletal muscle mass and function: Measurement and physiology of muscle fibre atrophy and muscle fibre loss in humans

“…However based on the current models used (pre-clinical models associated with genetic/irradiated ablation of SC; ( McCarthy et al, 2011 ; Rosenblatt et al, 1994 )) to assess the role of SC in skeletal muscle growth and ageing, and the continued conflicting findings arising from these experiments ( Blaauw and Reggiani, 2014 ), it remains unclear as to the importance of SCs in the maintenance of muscle in older age and in relation to muscle hypertrophy with RET. However, with new, novel stable isotope tracer techniques available for directly measuring both RNA and DNA synthesis in muscle ( Brook et al, 2017 ), the overall influence of ribosomal biogenesis and satellite cells in relation to anabolic resistance can also begin to be determined.…”

Quantifications of Lipid KineticsIn VivoUsing Stable Isotope Tracer Methodology

“…6C represents the time course changes in enrichments of precursor and product between 2 time points ( t 1 and t 2 ) where tissues (e.g., plasma) are typically harvested to measure changes in product enrichment and precursor enrichments for quantification of TAG FSR. The FSR term is also used in determining various polymers such as protein (from amino acids), 14 18 19 20 27 glucose (from 2 trioses), 28 29 DNA (from bases), 28 30 RNA (from bases), 31 32 and so on. In the following section, we will show flux calculation of selected variables of lipid kinetics using stable isotope tracer methodology and mass spectrometry.…”