Improving efficiency of protein deposition is one of the important goals in animal meat production. Theoretically as far as meat is involved, this efficiency can be increased by diminishing rate of muscle protein breakdown, provided that the rate of protein synthesis remains the same. In that case, the precursors needed for protein synthesis have to be made available by either decreasing the rate of oxidation of amino acids and/or by an increased intake of dietary protein. This implicates that waste of nitrogen into the environment would be reduced. In addition, less dietary protein is needed to deposit similar amounts of proteins in skeletal muscle (and thus meat) as in present situation. Moreover, by reduction of rate of protein breakdown, rate of protein turnover decreases, which reduces whole body energy requirement.
In order to manipulate the rate of muscle protein breakdown for interventions to increase efficiency of protein accretion, mechanisms and regulation of this process have to be known. However, the knowledge in this field is limited. The first approach of this thesis has been to study the relation between muscle protein breakdown in vivo and activity of proteolytic enzyme systems in skeletal muscle. The rate-limiting step in the cascade of muscle protein breakdown would be identified if activity of one of the proteolytic enzyme systems would change in parallel with a change in muscle protein breakdown. Thus, this would then open possibilities to manipulate efficiency of protein accretion.
The experiments presented in this thesis are carried out to investigate the relation between proteolytic enzyme systems and production of 3-methylhistidine (3MH). 3-Methylhistidine is a specific constituent of the myofibrillar protein, actin and myosin heavy chain. In most species, urinary excretion of 3MH can be used as an index for myofibrillar protein degradation, because this excretion is representative for the production of 3MH. In pigs, however, 3MH is not excreted in the urine, but is stored in skeletal muscle as a dipeptide called balenine (β-alanine-3-methylhistidine). In pigs, production rate of 3MH, rather than excretion rate, can be estimated using a compartmental model analysis. At the same time, three different proteolytic enzyme systems were studied in this thesis: the calpain system (μ- and m-calpain and their inhibitor calpastatin), multicatalytical proteinase and the lysosomal cathepsins, inhibited by the cystatins. As an experimental model, we have manipulated myofibrillar protein degradation by feeding a protein-free diet for 14 days to growing pigs.
In the first experiment (Chapter 2), relation between 3MH production and proteinase activities in different skeletal muscles was studied. Feeding a protein-free diet reduced growth rate to almost zero. The impaired muscle growth was also reflected by a reduced DNA transcription and translation. Production of 3MH was increased in the animals fed the protein-free diet, indicating that myofibrillar protein degradation was increased. However, no change was found in activities of one of the proteolytic enzyme systems in different skeletal muscles between treatments. This discrepancy can be explained in several ways. Firstly, production of 3MH may have been elevated form sources other than skeletal muscle. Secondly, other proteases are responsible for the ratelimiting step in the breakdown of myofibrillar proteins. Thirdly, proteinase assays performed in vitro may not represent physiological activity.
In the second experiment (Chapter 3), the findings of the first experiment were repeated (except for the lysosomal system). In addition, we examined the possibility that the observed findings by feeding a protein-free diet could be due to an increase in dietary carbohydrates. Therefore, a second protein-free diet was made by isocaloric exchange of dietary protein by fat. Half of the animals in each group were also realimentated after the protein-free feeding period for another 7 d, to investigate the relation between proteolytic enzyme systems and 3MH production during compensatory growth. No differences were found between treatments for 3MH production and for proteinase activities. Some compensation has only occurred during the first 3 d, based on data of growth rate and feed efficiency.
In Chapter 4, chemical composition of carcass, liver, and both large and small intestines were analyzed of the animals of the second experiment in order to study changes in fat and protein concentrations. Feeding either of the protein-free diets caused fattening of the carcass while protein concentration of carcass, liver, and intestines were decreased. After realimentation, protein concentrations were restored in liver and intestines, but not in the carcass. This suggests that protein metabolism in liver and intestines responds more rapidly to dietary changes than carcass.
In the third experiment (Chapter 5), the possibility that 3MH production was increased by sources other than skeletal muscle was investigated. The 3MH contribution from the gastro-intestinal tract was investigated by using two catheters, i.e. , placed in carotid artery and in portal vein. Results show that the 3MH contribution of gastro-intestinal tract to whole body 3MH production was not substantial and stayed below 5%. Moreover, feeding a protein-free diet did not change this contribution. Thus, increased 3MH production after feeding a protein-free diet originates mainly form increased breakdown of skeletal muscle.
In the fourth experiment (Chapter 6), an alternative approach was used to obtain more insight into the involvement of the calpain system during myofibrillar protein breakdown, since proteolytic activity is measured in vitro which may not reveal physiological activity. In this study, the calpain system was investigated at the transcriptional level by measuring mRNA levels. Results indicated that for m-calpain and calpastatin, mRNA levels correspond to proteolytic activity. For μ-calpain, the mRNA level was reduced on d 14 for animals in the protein- free group, though proteolytic activity was not different between dietary treatments. These data suggest that the calpain system is not involved in the rate-limiting step of myofibrillar protein degradation during a protein-free feeding period.
The general discussion (Chapter 7) describes the relation between 3- methylhistidine and proteolytic enzyme systems during a protein-free feeding period. The proteolytic enzyme systems examined do not seem to be responsible for the rate-limiting step during increased myofibrillar protein degradation as a consequence of feeding a protein-free diet. Therefore, other proteolytic enzyme systems must be responsible for this step. A model is discussed in which the degrading system does not necessarily contains the ratelimiting step. The rate-limiting step may be related to the dissociation of the myofibrillar structure itself.
|Qualification||Doctor of Philosophy|
|Award date||18 Jun 1996|
|Place of Publication||S.l.|
|Publication status||Published - 1996|