Measuring whole-body actin/myosin protein breakdown in mice using a primed constant stable isotope-infusion protocol

Abstract

To measure actin/myosin protein breakdown, the 24 h excretion of N (tau)-methylhistidine (3MH) is used. However, in mice, this method is invalid. Therefore we have developed a liquid chromatography-MS technique to measure the tracer/tracee ratio and concentration of 3MH in plasma, enabling an in vivo primed constant infusion protocol with a deuterated stable isotope of 3MH. We tested this model by giving a primed constant infusion of L-[3-methyl-(2)H(3)]histidine, L-[phenyl-(2)H(5)]phenylalanine and L-[phenyl-(2)H(2)]tyrosine to three anaesthetized experimental groups: mice receiving saline intraperitoneally (i.p.) (CON), mice receiving saline i.p. and starved for 9 h (STA), and mice receiving lipopolysaccharide i.p. and starved for 9 h (STA+LPS). The contribution of myofibrillar to total protein breakdown was significantly lower in the STA group than the CON group (30+/-4% and 54+/-14% respectively; P <0.05), and was significantly higher in the STA+LPS group than the STA group (52+/-7% and 30+/-4% respectively; P <0.05). Whole-body myofibrillar protein breakdown, total protein breakdown, protein synthesis and net protein breakdown were not different between the groups. We conclude that this in vivo primed constant stable isotope-infusion protocol can give valuable information about the role of actin/myosin protein breakdown in mice.

Time course of nitric oxide production after endotoxin challenge in mice

“…Plasma Phe levels and Phe flux during endotoxemia. In contrast to Arg, Cit, and Gln, arterial Phe levels were increased in the early phase of sepsis, which is in agreement with previous studies in mice that showed increased plasma Phe values at 6 h after LPS administration (16) but no increments at 11 or 19 h (14,38). In septic patients, Phe was shown to be the only amino acid whose plasma concentrations were increased (12).…”

Effects of Peripartum Propylene Glycol Supplementation on Nitrogen Metabolism, Body Composition, and Gene Expression for the Major Protein Degradation Pathways in Skeletal Muscle in Dairy Cows

“…Whole-body N balance, body protein content, urinary 3-MH excretion, and transcript abundance profiles for the major protein degradation systems were used as gross indices of body protein metabolism. Urinary excretion of 3-MH is a commonly used indicator of the extent of myofibrillar protein degradation (Vissers et al, 2003), and an elevation in urinary 3-MH excretion has been taken to be indicative of an increase in the breakdown of myofibers in skeletal muscle (Plaizier et al, 2000b). Various protein degradation systems in skeletal muscle have been characterized, including the lysosomal system, the caspase system, the Ca 2+ -dependent system, and the ubiquitin-mediated, ATP-dependent system.…”

Measuring muscle protein turnover in vivo: what can 3-methylhistidine production tell us?

“…The paper by Vissers et al [16] in this issue of Clinical Science describes a refinement of the 3MH technique for use in mice. It is known that 3MH excretion cannot be measured satisfactorily in mice, because some of the 3MH is excreted as conjugated metabolites (although these can be released by hydrolysis) and some is temporarily sequestered in to unidentified pools.…”

“…It will be interesting to see what use can be made of this technique [16]. Recent advances in proteomics are showing that huge numbers of different proteins exist in different tissues [19], so that data on the average rate of protein turnover even at the level of the whole tissue are likely to mask important differences between individual proteins.…”

“…It is known that 3MH excretion cannot be measured satisfactorily in mice, because some of the 3MH is excreted as conjugated metabolites (although these can be released by hydrolysis) and some is temporarily sequestered in to unidentified pools. Hence Vissers et al [16] have developed a method for measuring 3MH production directly. This has the additional advantage of being sensitive to changes over a shorter timescale than the 24 h over which urine collections are conventionally made.…”