Existence of life-time stable proteins in mature rats—Dating of proteins’ age by repeated short-term exposure to labeled amino acids throughout age

PLOS ONE, Nov 2019

In vivo turnover rates of proteins covering the processes of protein synthesis and breakdown rates have been measured in many tissues and protein pools using various techniques. Connective tissue and collagen protein turnover is of specific interest since existing results are rather diverging. The aim of this study is to investigate whether we can verify the presence of protein pools within the same tissue with very distinct turnover rates over the life-span of rats with special focus on connective tissue. Male and female Lewis rats (n = 35) were injected with five different isotopically labeled amino acids tracers. The tracers were injected during fetal development (Day -10 to -2), after birth (Day 5–9), at weaning (Day 25–32) at puberty (Day 54–58) and at adulthood (Day 447–445). Subgroups of rats were euthanized three days after every injection period, at different time point between injection periods and lastly at day 472. Tissue (liver, muscle, eye lens and patellar tendon) and blood samples were collected after euthanization. The enrichment of the labeled amino acids in the tissue or blood samples was measured using GC-MS-MS. In muscle and liver we demonstrated a rapid decrease of tracer enrichments throughout the rat’s life, indicating that myofibrillar and cytoskeleton proteins have a high turnover. In contrast, the connective tissue protein in the eye lens and patellar tendon of the mature rat showed detainment of tracer enrichment injected during fetal development and first living days, indicating very slow turnover. The data support the hypothesis that some proteins synthesized during the early development and growth still exist much later in life of animals and hence has a very slow turnover rate.

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Existence of life-time stable proteins in mature rats—Dating of proteins’ age by repeated short-term exposure to labeled amino acids throughout age

September Existence of life-time stable proteins in mature ratsÐDating of proteins' age by repeated short-term exposure to labeled amino acids throughout age Cecilie Leidesdorff Bechshøft 1 2 3 Peter Schjerling 1 2 3 Andreas Bornø 0 1 3 Lars Holm 0 1 2 3 0 Clinical Metabolomics Core Facility, Department of Clinical Biochemistry , Rigshospitalet, Copenhagen , Denmark , 3 Institute of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark 1 Funding: Danish Research Council for Independent Research, Health and Disease, grant number:09- 073587 to Mr Lars Holm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Institute of Sports Medicine, Department of Orthopedic Surgery M, Bispebjerg Hospital and Center of Healthy Aging, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark 3 Editor: Jon M. Jacobs, Pacific Northwest National Laboratory , UNITED STATES In vivo turnover rates of proteins covering the processes of protein synthesis and breakdown rates have been measured in many tissues and protein pools using various techniques. Connective tissue and collagen protein turnover is of specific interest since existing results are rather diverging. The aim of this study is to investigate whether we can verify the presence of protein pools within the same tissue with very distinct turnover rates over the lifespan of rats with special focus on connective tissue. Male and female Lewis rats (n = 35) were injected with five different isotopically labeled amino acids tracers. The tracers were injected during fetal development (Day -10 to -2), after birth (Day 5±9), at weaning (Day 25± 32) at puberty (Day 54±58) and at adulthood (Day 447±445). Subgroups of rats were euthanized three days after every injection period, at different time point between injection periods and lastly at day 472. Tissue (liver, muscle, eye lens and patellar tendon) and blood samples were collected after euthanization. The enrichment of the labeled amino acids in the tissue or blood samples was measured using GC-MS-MS. In muscle and liver we demonstrated a rapid decrease of tracer enrichments throughout the rat's life, indicating that myofibrillar and cytoskeleton proteins have a high turnover. In contrast, the connective tissue protein in the eye lens and patellar tendon of the mature rat showed detainment of tracer enrichment injected during fetal development and first living days, indicating very slow turnover. The data support the hypothesis that some proteins synthesized during the early development and growth still exist much later in life of animals and hence has a very slow turnover rate. Data Availability Statement; All relevant data are within the paper Introduction To allow growth and remodeling as well as functional integrity most proteins are undergoing a process of continuous turnover involving degradation of existing proteins and biosynthesis of replacement proteins. Synthesis and breakdown rates of proteins have been measured in many tissues and protein pools using various techniques and half-lives ranging from few hours to months/years have been found in larger animals and humans. Connective tissue and collagen protein turnover is of specific interest, since existing results on synthesis (and turnover) rates are conflicting. Some studies indicate that matrix collagen proteins synthesized early in life in humans are preserved into adulthood in tissues like eye lenses and tendon [ 1, 2 ]. In contrast, other studies utilizing other techniques report that collagen protein in human tendon and muscle has a synthesis rate in adulthood [3] and in older age [ 4, 5 ] that is comparable to e.g. myofibrillar proteins from skeletal muscle. Specifically in relation to collagen protein the turnover rates can be determined by utilizing different methodological approaches and principles. For example, several possibilities exist to measure changes in concentrations of biomarkers referring either to the synthesis or breakdown processes of collagen. One is the posttranslational hydroxylation of proline residues to hydroxyproline in collagen. Since hydroxyproline is not reutilized for protein synthesis it is disposed to the plasma and excreted in the urine upon degradation of collagen proteins [6]. Therefore, the amount of hydroxyproline in the blood and/or urine is used to reflect collagen protein degradation [ 6, 7 ]. Similarly, abundance of hydroxyproline with microdialysis technique has been demonstrated in human skeletal muscle [ 8 ] and thereby collagen degradation has been shown. Other types of assessments of collagen protein turnover involves the measurement of abundances of biomarkers such as pro-collagen type I C-terminal peptide (PICP), pro-collagen type I N-terminal peptide (PINP), COOH-terminal telopeptides of type-I collagen (CTX-I) and Cterminal type II pro-colla (...truncated)


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Cecilie Leidesdorff Bechshøft, Peter Schjerling, Andreas Bornø, Lars Holm. Existence of life-time stable proteins in mature rats—Dating of proteins’ age by repeated short-term exposure to labeled amino acids throughout age, PLOS ONE, 2017, Volume 12, Issue 9, DOI: 10.1371/journal.pone.0185605