The Limiting Amino Acid Concept and Modelling

It is not clear whether the relationship between AA supply and protein synthesis is simply a substrate effect or a reflection of regulatory events. Although acyl-tRNA are normally saturated in other tissues at prevailing intracellular AA concentrations (Shenoy and Rogers, 1978), the same does not appear to be the case for the udder (Elska et ai, 1971). If the tRNA-acylating enzymes are not saturated with AA under normal conditions, then provision of additional AA should result in an increase in acylated-tRNA concentrations and more efficient rates of mRNA translation. Given that all 20 AA are required to synthesize milk protein, limitations may occur simultaneously for any of a number of amino acylated-tRNA, e.g. when an AA is at less than saturating concentrations the ribosome may 'hesitate' slightly at each codon specifying the AA. Consequently, the relative deficiency and the number of moles of that particular AA required to synthesize a mole of milk protein would determine the substrate response when the deficiency is alleviated. The implications are that multiple AA may be rate limiting for milk protein synthesis at any one time. This is at odds with the traditional definition and use of the term 'nutritionally limiting AA' where only one AA can be limiting. The multiple AA concept is consistent with the observations by Clark et al. (1978), wherein responses to three different AAs were observed under identical culture conditions. If milk protein synthesis is sensitive to multiple AA at the same time, then changes in arterial concentrations of each AA could be very important when predictions of milk protein production are attempted.

There are other points of regulation of protein synthesis that may need to be considered in refining existing models. The initiation step of protein synthesis is regulated by a variety of factors, including leucine, alanine, gluta-mine and histidine (Yokogoshi and Yoshida, 1980; Perez-Sala et ai, 1991) and their unacylated-tRNAs (Iiboshi et al., 1999). Leucine exerts control of translation via intracellular signalling mechanisms that facilitate more efficient translation of mRNA. These pathways are common to those regulated by insulin. In some cell models and in vivo, the branched-chain AA, leucine specifically, enhances tissue sensitivity to insulin (see Jefferson and Kimball, 2001). This role for leucine appears to be permissive, however, because infusion of the branched-chain AA in dairy cows either alone or in combination with infusion of insulin (hyperinsuli-naemic-euglycaemic clamp) does not result in a further enhancement of milk protein yield (Annen et al., 1998; Mackle et al., 1999). Volume regulated control of cellular protein synthesis (the cell swelling hypothesis) by glutamate may also be an important mechanism in global and milk protein synthesis as demonstrated by Millar et al. (1997) in rat mammary acini. Peptide chain elongation and termination, and expression and turnover of milk protein mRNA, are also potential points of regulation. These points of control have yet to be examined in mammary epithelial cells. If these systems operate in mammary tissue, then AA may also act as direct regulators of casein synthesis. Given the complexity of the system, it is not surprising that progress has been slow using empirical (feeding or infusion studies) approaches to define requirements for individual AA.

Despite the apparent complexity, milk protein synthesis has typically been represented as a simple linear function of the most limiting AA (Hanigan and Baldwin, 1994;

Maas et al., 1997) or of AA supply in aggregate (Baldwin et al., 1987; Danfaer, 1990) as has been adopted for monogastric growth models (D'Mello, 1994). However, the observations by Clark et al. (1978) suggest that such a simple representation may not be adequate for mammary tissue. Hanigan et al. (2002a) devised an equation that may be more representative, given the previous discussion:


AA,Pm fk \ExPni,Pm ni.Pm

m where UAAPm represents the conversion of AA into milk protein, V^^ represents the maximal velocity of the reaction, Cni represents the intracellular concentration of individual AA (1 to n), kni>Pm represents the apparent affinity constants for the respective AA considered in the representation, and Expni Pm represents an exponent that can be used to adjust sensitivity to concentrations of individual AA, if needed. Adjustment of the various exponents to values other than 1 should reflect the varied molar proportions of each AA in milk protein and the relative importance of the respective AA in the regulatory process. Equation [19.1] was found to provide slightly more accurate predictions of milk protein yield when used to simulate 21 da ta sets from the literature, compared to an equation based on the single-limiting AA theory. However, many of the experiments used in the analysis involved treatments where all AA were manipulated simultaneously, i.e. casein infusions. Consequently, the hypothesis that individual AA independently affect milk protein synthesis was not well tested, other than for lysine and methionine. Visual appraisal of the methionine and lysine infusion work indicated that the model fitted those observations significantly better. Use of this multisubstrate representation and a representation of the transporter system that included intracellular feedback control of transporter activity (Hanigan et al., 2001a,b, 2002), helped to explain data where the udder was able to minimize a 20% drop in milk protein production when arterial histidine concentrations declined ninefold (Bequette et ai, 2000).

The above prediction scheme is driven by an innate drive to produce milk, e.g. VM Pm. This drive is tempered by endocrine control and the ability to maintain intracellular concentrations of AA. As such, accurate predictions of intracellular AA concentrations are important. Assuming milk protein synthesis and AA removal can be accurately represented by a combination of equations, one need only define the relationship describing AA catabolism in order to predict concentrations. This assumes that the udder is not growing and that non-free sources of AA are insignificant contributors to AA supply, which for the latter may not be true (see above). The various mammary models that have attempted to describe intracellular AA concentrations (Waghorn and Baldwin, 1984; Hanigan and Baldwin, 1994; Maas et al, 1997; Hanigan et al, 2001b) have made the general assumption that AA catabolism is a mass action function of AA concentration. So far, this assumption appears to be correct for leucine, phenylalanine and lysine (Bequette et al, 1996a,b, 1999, 2002; Mabjeesh et al, 2000). However, if the regulation of mammary AA oxidative enzymes is more complicated than simple substrate interactions (i.e. physiological changes in gene expression; DeSantiago et al, 1998), then a more complex representation may be required.

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