Muscle free amino acid profiles are related to differences in skeletal muscle growth between single and twin ovine fetuses near term

Increasing prolificacy is an effective way to improve profitability in sheep production systems (Gootwine et al. 2001). However, birth weight, postnatal survival, growth, body composition and lifetime production performance may be reduced as litter size increases (Barker 1998; Greenwood et al. 1998; Morel et al. 2009). Reduced fetal weight near term in twins compared to singletons is associated with decreased skeletal muscle hypertrophy, leading to reduced muscle mass (McCoard et al. 2001). Although maternal undernutrition has a direct effect on fetal and skeletal muscle growth during gestation (Fahey et al. 2005), reduced fetal weight and muscle weight in twins compared to singles is observed even in well-nourished ewes (Freetly and Leymaster 2004). This suggests maternal nutrition is not the only factor to impact fetal and muscle growth as litter size increases.

It is well established that fetal growth is influenced by fetal amino acid (AA) availability (De Boo et al. 2005; Kwon et al. 2004; Liechty et al. 1999). Studies in sheep indicate that the rate of protein accretion in the fetus can be stimulated through the fetal infusion of a mix of AA (De Boo et al. 2005; Liechty et al. 1999). Importantly, AA also have the capacity to signal to metabolic pathways which regulate muscle growth (Brown et al. 2009; Hara et al. 1998) via changes in the intracellular concentration of specific AA (Beugnet et al. 2003; Christie et al. 2002; Sancak et al. 2008). For example, AA signalling plays an important role in the regulation of skeletal muscle hypertrophy in monogastrics, through the activation of specific cell signalling pathways (e.g. mechanistic target of rapamycin, mTOR), which controls protein synthesis (Tan et al. 2009; Yao et al. 2008). However, the potential for specific AA to act as signalling molecules to regulate skeletal muscle hypertrophy during gestation in ruminants is not well understood.

In sheep, we have preliminary evidence (Pacheco et al. 2010) that concentrations of specific intracellular AA in skeletal muscle (e.g. arginine and glutamine), differ between single and twin fetuses in late gestation in nutritionally-restricted ewes. The purpose of this study was to further explore the potential relationship between skeletal muscle mass and intracellular free AA concentration in twins compared to singletons, by testing two hypotheses. Our first hypothesis is that reduced skeletal muscle weight in twin, compared to single fetuses in late gestation, is associated with reduced concentration of specific free AA in muscle, such as glutamine and arginine. Our second hypothesis is, that dam nutrition and body size influence the relationship between skeletal muscle AA and fetal muscle weight, between pregnancy ranks (single vs. twin). To test these hypotheses, the concentration of free AA from the semitendinosus (ST) muscle collected from twin and singleton fetuses at 140 days of gestation from heavy and light ewes fed two differing planes of nutrition were compared.

A three-way interaction between pregnancy rank, size and nutrition was observed for ST weight (P = 0.04, Figure 1). Twins from HM-ewes had lower ST weights compared with twins from HA-ewes, whereas singletons from LM-ewes had lower ST weight compared with LA singletons (Figure 1). After adjusting for fetal body weight, only a pregnancy rank effect was observed, whereby twins had 17% lower ST weight compared to singles (8.3 ± 0.3 g vs. 10.0 ± 0.3 g, P = 0.002). No maternal size, maternal nutrition or interactions with maternal size or nutrition effects were observed for ST weight (data not shown).

A pregnancy rank by nutrition interaction was observed for alanine and aspartic acid concentration in ST muscle (Table 2). Twin fetuses had 13% lower (P = 0.02) alanine concentration compared to singletons in the M-ewes, while no difference was observed between pregnancy ranks in the A-ewes. For aspartic acid, twin fetuses from A-ewes had 33% lower (P < 0.0001) concentration compared to singletons from A-ewes, while no difference was observed between ranks in the M-ewes group.

Compared to singletons, twins had lower ST muscle concentration of leucine (19%), threonine (23%), valine (36%) and total EAA (11%) (Table 2), but higher concentrations of methionine (67%), ornithine (19%), lysine (23%), serine (33%) and total non EAA (5%) (Table 2).

A positive association was observed between ST muscle weight and arginine concentration after partial correlation analysis (Figure 2). In contrast, a negative correlation was found between ST muscle weight and intracellular concentration of taurine (Figure 3). No correlations were found for any other FAA with ST muscle weight (data not shown).

The objective of this study was to explore the potential relationship between skeletal muscle weight and intracellular FAA concentration in single compared to twin fetuses in late gestation sheep. An additional objective of this study was to establish the effect of maternal size and nutrition on fetal muscle FAA concentration, as an approach to understand some of the possible mechanisms explaining the lower muscle mass normally observed in twin fetuses (McCoard et al. 2001). Reduced ST mass in twins compared to singleton fetuses at 140 days gestation was associated with changes in the concentration of specific but not total intracellular FAA concentration. Notably, the concentrations of arginine, leucine, valine, and glutamine, known to influence pathways which regulate protein synthesis (Wu 2009), were lower in muscle of twin compared to single fetuses. While other AA were affected by pregnancy rank, maternal size or nutrition, their role in fetal muscle growth, beyond being the building blocks for protein synthesis, is unclear. Arginine concentration, irrespectively of pregnancy rank, maternal size and maternal nutrition, was the only AA positively correlated with fetal skeletal muscle mass. These results suggest that arginine may be important for skeletal muscle growth in the late-gestation ovine fetus.

Irrespective of maternal size or maternal nutrition, twin fetuses had lower ST muscle mass compared to singletons, in agreement with a previous study (McCoard et al. 2001). In the present study, the effect of maternal nutrition on muscle mass was influenced by maternal size. Twin fetuses from HM-ewes had disproportionately smaller ST muscle compared to their single counterparts. This indicates that heavy twin-bearing ewes fed a maintenance plane of nutrition were unable to meet the nutritional requirements to maintain not only fetal weight (Blair et al. 2011), but also muscle growth, when compared to their counterparts carrying singletons. Competition for limited nutrients between twins (McCoard et al. 2000) or the reduced priority of nutrient partitioning to fetal skeletal muscle development compared with other organs during maternal nutrient restriction (Du et al. 2010) could explain the lower muscle mass in twins from ewes at a restricted feeding level. In contrast, the lower ST muscle weight observed in singles and twins from LM-ewes, in comparison with their single and twin counterpart from LA-ewes, could indicate that lighter ewes fed a maintenance level of nutrition are unable to provide the nutrient requirements either for a single or twin pregnancy. These results support the notion that skeletal muscle growth in twins is more sensitive to maternal nutritional constraint than in singles, which is in agreement with a previous study (Gootwine et al. 2007). However, our results also suggest muscle growth can be compromised in singleton pregnancies under maternal nutrient restriction, as reported previously (Quigley et al. 2008).

Free AA plays a major role not only as building blocks for protein synthesis, but they also regulate key metabolic pathways which are necessary for cell maintenance and growth (Wu 2009). The function of FAA as signalling molecules is associated with changes in the intracellular concentration of specific FAA (Beugnet et al. 2003; Christie et al. 2002; Sancak et al. 2008). Intracellular pools are therefore critical to accomplish the signalling function of FAA (Nobukuni et al. 2005). The size and composition of the intracellular FAA pool depends on different processes, including availability of circulating FAA, and an increased AA influx or efflux between muscle and the plasma resulting from utilization, (e.g., by protein synthesis) or catabolism (protein turnover) (Hundal and Taylor 2009; Proud 2004). In this study it is unclear what the contribution is of each of these processes to the observed differences in intracellular FAA profiles. Unfortunately, fetal plasma was not available in the present study to relate the plasma FAA profile with intracellular muscle FAA profile in single and twin fetuses. However, we have previously shown that twins from ad libitum-fed ewes have lower plasma concentration of glutamine, arginine and leucine compared with singletons at day 140 of pregnancy (van der Linden et al. 2012). Others have also reported a decreased concentration of arginine family members and branched-chain amino acids (BCAA) in fetal plasma when restricting maternal nutrition to 50% of their requirement (Kwon et al. 2004). It has also been proposed that reduced muscle mass in sheep fetuses exposed to maternal nutrient restriction may be associated with reduced plasma FAA, particularly serine, arginine-family AA, and BCAA (Zhu et al. 2006). Therefore, it is feasible that changes in specific FAA concentration in twins muscle were related to changes in circulating AA availability. The possible association between pregnancy rank and maternal nutrition on fetal plasma AA concentration and how this affects intracellular muscle FAA concentrations and muscle mass, is yet to be established.

Skeletal muscle growth in fetuses utilizes both EAA and non-essential AA (NEAA) (Wilkening et al. 1994). However, during fetal stress, such as maternal fasting, AA catabolism increases in muscle, resulting in release to the circulation of gluconeogenic precursors such as glutamine (Liechty and Lemons 1984), due to an increased metabolism of BCAA (Liechty et al. 1987). In the present study, the reduced concentration of the BCAA leucine and valine in ST muscle of twins compared to singletons could suggest higher muscle protein breakdown in twins, resulting in a lower muscle mass compared to singles. In addition, protein breakdown may have also contributed to the observed reduction in glutamine concentration in ST muscle of twin fetuses from HM-ewes. This may have led to the higher growth limitation of muscle from fetuses in the HM group, reinforcing the idea of a greater degree of restriction in this group. Whether the difference in intracellular concentration of FAA is related to changes in FAA transport or a result of catabolism, warrants further investigation.

Arginine is considered a conditionally indispensable AA for the fetus (Wu et al. 2009) and participates in the synthesis of proteins, nitric oxide, polyamines, creatine, some AA and agmatine (Wu and Morris 1998), playing a major role in skeletal muscle growth (Wu et al. 2000). The exacerbated reduction in arginine concentration observed in twins compared to singles from H-ewes compared to their L-ewes counterparts, may be associated with a more stressful fetal environment. Reduced concentration of arginine family members as a result of maternal undernutrition, has been previously described in fetal plasma (Kwon et al. 2004) and in gastrocnemius muscle of sheep fetuses (Wu et al. 2006), which supports our findings. The reduced concentration of arginine, could be due to a higher catabolism of this AA, which would explain the increase in ornithine concentration observed in twins compared with singletons, according to Wu and Morris (1998). The higher concentration of arginine found in singletons compared to twins and the positive, although weak partial correlation between intracellular muscle arginine with muscle mass, suggests that arginine may act as a limiting AA for fetal muscle growth in twin fetuses.

Protein synthesis in muscle is controlled by specific signalling pathways, including phosphatidylinositol-3 kinase (Schiaffino and Mammucari 2011), 5′-AMP-activated protein kinase (Bolster et al. 2002) and mitogen-activated protein kinase (Williamson et al. 2003). However, mTOR is accepted as the major pathway regulating muscle protein synthesis (Du et al. 2005). Intracellular FAA can activate mTOR (Beugnet et al. 2003), whereas a decrease in the intracellular AA concentration reduces the mTOR signalling (Sancak et al. 2008). Specific AA such as leucine (Escobar et al. 2006; Suryawan et al. 2008; Suryawan et al. 2011) and arginine (Yao et al. 2008) activate mTOR in muscle of monogastrics, as well as glutamine in cell culture models (Chiu et al. 2012; Nicklin et al. 2009). Preliminary evidence indicates a reduced abundance of mTOR downstream targets in muscle of twin fetuses, compared with singletons at late gestation (Sciascia et al. 2010). Therefore, it is possible that reduced intracellular concentrations of glutamine, leucine and arginine in muscle of twins compared to singles in this study, could have resulted in a decreased mTOR signalling, and therefore, reduced muscle mass. This potential mechanism is part of future research.

The increase in methionine concentration in ST muscle from twins compared to singles is a novel finding. Methionine is an EAA, used to initiate protein synthesis (Kozak 1983); it is involved in DNA methylation (Waterland 2006), participates in oxidative processes (Hoshi and Heinemann 2001) and has other metabolic functions (Brosnan and Brosnan 2006). Previous studies in rats under starvation have shown the increased concentration of methionine in muscle as a result from high protein breakdown (Millward et al. 1974; Millward 1970). However, reduced utilisation from lower protein accretion cannot be excluded.

Reduced concentrations of specific FAA, which are known to play a role in muscle growth, could act as limiting factors for muscle hypertrophy in twin fetuses in late gestation. The effect of decreased concentration of leucine, valine, glutamine, and especially arginine on fetal skeletal muscle growth in late gestation requires further investigation. The consequences of a maintenance maternal nutrition and maternal weight on fetal muscle mass and concentration of some FAA, reinforces their importance for fetal muscle growth. Altogether, these findings establish a baseline for new studies to further define the role of AA in fetal muscle growth in sheep, and open new possibilities for future strategic nutritional interventions to improve skeletal muscle development.