Leu is recognized as a signaling factor for translation initiation and MPS [5, 18, 20]. However, the majority of this research has been done with large acute doses of free Leu, and the significance of this signal in physiological meals with different dietary proteins remains largely unknown. Accordingly, the present study examined responses of plasma amino acids, translation initiation factors, and MPS in adult rats fed complete meals with protein sources selected for differences in Leu content. This study demonstrated that in small meals with limited protein intake, MPS was stimulated in proportion to activation of translation factors and further establishes a threshold requirement for a minimum Leu content within a meal.
The relationship between post-prandial plasma Leu concentrations and stimulation of MPS has been previously observed with both animal [7, 21] and human [22, 23] studies. However, other studies reported using supplemental Leu producing substantial increases in plasma Leu that had no incremental effect on MPS [24–26]. While these studies appear contradictory, taken together, these studies are consistent with the hypothesis that Leu acts as a threshold trigger for activation of translation initiation. As plasma Leu increases after a meal from reduced fasted concentrations to post-prandial peak values, the change in concentration is recognized by the mTORC1 signal complex and triggers initiation. Once the threshold for activation has been achieved and the active ribosome complex assembled, additional increments in plasma Leu have minimal effects [5, 7].
Norton et al. conducted a dose response study examining wheat and whey proteins fed at 10%, 20% and 30% of dietary energy. At isonitrogenous intakes, whey protein produced higher levels of plasma Leu, mTOR signaling, and MPS than wheat. However, MPS was not different comparing meals with 30% wheat vs. 20% whey consistent with the net Leu available from wheat (6.8% Leu) and whey (10.9% Leu). Similar support for Leu as a signal to initiate an anabolic meal response has been observed for young men consuming whey, soy and casein after exercise  and in older men or rats consuming whey versus casein [21, 22].
To further test the physiological significance of the threshold hypothesis, Experiment 1 utilized a meal-feeding protocol with adult rats. Based on previous research with rats of the same age and size  we estimated that the Leu required to achieve the initiation signal threshold at a meal would be ~50 to 60 mg of Leu. We selected 4 proteins that provided a range of Leu contents and designed a meal to provide 20% of daily energy and 16% of energy as protein. This combination of meal size and protein content allowed the 4 proteins to bracket the proposed Leu threshold (ie. wheat 46 mg, soy 54 mg, egg 60 mg, and whey 74 mg of Leu). The whey and egg proteins provided sufficient Leu to increase plasma Leu concentrations, increase phosphorylation of translation factors S6K1 and 4E-BP1, and stimulate MPS. These findings support a concept for a minimum meal threshold for Leu to stimulate post-prandial MPS.
While dietary Leu appears to be a critical factor to account for the post-meal MPS response, this does not exclude potential contributions from other amino acids. There is evidence that orally administered isoleucine (Ile) also activates mTORC1 signaling, although not as potent as Leu . In complete proteins, Ile is typically present in proportion to the Leu content, allowing the possibility that Ile may in part contribute to the mTORC1 signaling and MPS. However, in the current study, rats fed soy significantly increased post-prandial plasma Ile concentration with no corresponding increase in mTORC1 signaling or MPS. This is consistent with previous research that only Leu among the EAA was able to increase MPS when infused in rats . In the present study with complete meals, only the post-prandial changes in plasma Leu predicted changes in mTORC1 signaling and stimulation of MPS.
Beyond Leu and Ile contents, dietary proteins differ in other characteristics including distribution of other indispensable amino acids, differences in gastric emptying or digestibility, and insulinogenic properties. Experiment 2 was designed to isolate the importance of Leu within a meal and to test the significance of Leu content versus other intrinsic properties of protein sources. Experiment 2 demonstrated that supplementing the wheat protein meal with Leu to equalize the Leu content of the meals (wheat + Leu = whey) produced similar post-prandial patterns for plasma Leu and MPS response. Further, for each of the treatment groups, peak plasma Leu concentration occurred 30 min after the meal, and there was a strong correlation with Leu content of the meal and peak plasma Leu concentration (r = 0.919; P < 0.05). These findings are consistent with the post-exercise data of Churchward-Venne et al.  demonstrating that a suboptimal dose of whey protein (6.25 g) supplemented with Leu could produce the same MPS rate as a high dose of whey protein (25 g). These studies confirm that between different protein sources Leu content is an important predictor of post-meal MPS responses.
Also supporting the Leu threshold hypothesis, there are numerous examples of saturation of the Leu signal [5, 7, 19]. Using oral doses of free Leu ranging from 0.068 g/kg body weight up to 1.35 g/kg, Crozier et al.  demonstrated that the maximum rate of MPS was achieved in a 200 g rat at ~67 mg of Leu and that this represented maximum activation of translation initiation signals. Increasing dosage of Leu to double and triple this amount proportionally increased plasma Leu concentration but had no additional effects on initiation factors or MPS. Likewise, Debras et al.  observed no difference in MPS after a casein-based meal containing 81 mg of Leu versus casein plus Leu supplement with 237 mg of Leu; Norton et al.  found that a meal with 30% of energy from wheat gluten was similar to a 20% whey protein meal in producing maximum MPS and translation initiation responses; and Anthony et al.  reported that rats fed either a 20% soy or 20% whey protein meal after exercise both increased plasma Leu to approximately double baseline concentrations and both diets fully stimulated mTORC1 signaling and MPS. In total, these studies demonstrate that Leu is an essential threshold signal for translation initiation but additional amounts above the threshold do not produce additive effects on activation.
The current studies demonstrate that insulin does not differentiate regulation of post-prandial MPS in adult rats consuming that same amounts of total carbohydrates and protein. Prior research suggests that insulin plays a permissive role in Leu-dependent stimulation of protein synthesis and translation initiation . Consistent with this view, all protein groups in the current studies exhibited post-prandial increases in insulin and/or the down-stream signal element Akt-phosphorylation allowing for a permissive anabolic response to the meal, however the precise insulin concentration or the activation state of Akt had no apparent relationship to absolute rates of MPS. In the current studies, the post-prandial insulin response was relatively small presumably due to meal-feeding protocol and restricted carbohydrate load of the test meals. In Experiment 2, the insulin response was greatest 30 min post-meal in the whey and wheat + Leu groups relative to baseline control and wheat groups consistent with increases in MPS. While the test meals contained equal quantity and quality of carbohydrates and equal amounts of protein, the heightened insulin response in whey protein and wheat + Leu fed rats may contribute to greater rates of mTOR signaling and MPS. The specific contribution of insulin to MPS regulation in adults remains to be fully elucidated.