Factors affecting dietary protein-derived amino acid release into the circulation

The combination of intrinsically labelled protein with intravenous stable isotope amino acid infusion has been applied to identify different dietary factors that modulate EXO_Ra. The ingestion of greater amounts of protein results in a greater EXO_Ra up to (at least) the ingestion of as much as 45 g protein^{(Reference Churchward-Venne, Pinckaers and Smeets6,Reference Holwerda, Paulussen and Overkamp25–Reference Kouw, Holwerda and Trommelen27)} . Different types of proteins also modulate EXO_Ra. For instance, casein protein ingestion results in a relatively lower but more prolonged EXO_Ra, whereas whey protein ingestion results in a relatively rapid, but more transient, increase in EXO_Ra^{(Reference Pennings, Boirie and Senden8,Reference Boirie, Dangin and Gachon16,Reference Gorissen, Trommelen and Kouw26)} . This difference has been attributed to the clotting of micellar casein protein in the acidic environment of the stomach, thereby delaying gastric emptying and subsequent protein digestion^{(Reference Ye, Cui and Dalgleish28)}. Milk protein ingestion results in an intermediate EXO_Ra when compared to whey and casein protein ingestion^{(Reference Gorissen, Trommelen and Kouw26)}. Milk protein ingestion is followed by a lower EXO_Ra when compared to the ingestion of an equivalent amount of minced (cooked) beef^{(Reference Burd, Gorissen and van Vliet29)}. The digestion and absorption kinetics of ingested protein are not only dependent on the inherent properties of the protein, but are also modulated by protein processing and/or meal preparation. In this regard, the ingestion of a protein hydrolysate results in an accelerated EXO_Ra when compared with the ingestion of the same, intact protein^{(Reference Pennings, Boirie and Senden8,Reference Koopman, Crombach and Gijsen30)} . In a similar context, the ingestion of minced (cooked) meat results in a higher EXO_Ra when compared to the ingestion of (cooked) beef steak^{(Reference Pennings, Groen and van Dijk31)}. Furthermore, co-ingestion of other macronutrients impacts the EXO_Ra following protein ingestion. For instance, carbohydrate co-ingestion attenuates the EXO_Ra^{(Reference Gorissen, Burd and Hamer32)}. In agreement, the EXO_Ra of casein is attenuated when ingested within a carbohydrate-containing milk matrix^{(Reference Churchward-Venne, Snijders and Linkens33)}. However, the co-ingestion of milk fat within a casein protein beverage does not impact EXO_Ra^{(Reference Gorissen, Burd and Kramer34)}. In contrast, egg-white consumption resulted in a more rapid EXO_Ra when compared to the ingestion of an isonitrogenous amount of whole egg that contained substantially more fat^{(Reference van Vliet, Shy and Abou Sawan35)}. The discrepancy in the impact of fat co-ingestion on EXO_Ra is possibly due to how fat interacts with protein within the food matrix. For instance, co-ingested fat likely separates from protein within the stomach following consumption as a beverage, whereas fat and proteins may remain homogenous in a matrix following whole egg ingestion, facilitating greater macronutrient interaction^{(Reference Edelbroek, Horowitz and Maddox36)}.

In addition to dietary factors, the EXO_Ra is modulated by physiological conditions. Protein ingestion in older individuals results in lower EXO_Ra when compared to the ingestion of the same amount of protein by younger adults^{(Reference Gorissen, Trommelen and Kouw26,Reference Gorissen, Burd and Hamer32,Reference Groen, Horstman and Hamer37)} , and EXO_Ra is lower in obese v. lean individuals^{(Reference Kouw, van Dijk and Horstman38)}. Furthermore, EXO_Ra seems to be attenuated during recovery from resistance-, but not endurance-type exercise^{(Reference van Wijck, Pennings and van Bijnen39,Reference Mazzulla, Parel and Beals40)} . Finally, maintenance haemodialysis patients display a lower EXO_Ra following egg ingestion when compared to healthy controls^{(Reference van Vliet, Skinner and Beals41)}. Taken together, it is clear that dietary protein-derived amino acid release is strongly modulated by several factors related to the composition and preparation of the ingested meal along with other physiological factors.

Factors affecting post-prandial whole-body protein metabolism

The combined application of continuous intravenous infusion of stable isotope amino acids and ingestion of intrinsically labelled protein has been used to establish that intrinsically labelled milk protein ingestion stimulates whole-body protein synthesis rates, attenuates whole-body protein breakdown rates and improves whole-body protein net balance in a dose-dependent manner up to doses of at least 45 g^{(Reference Churchward-Venne, Pinckaers and Smeets6,Reference Holwerda, Paulussen and Overkamp25,Reference Kouw, Holwerda and Trommelen27)} . This response can be modulated by the type of protein ingested. For example, the ingestion of intrinsically labelled minced beef has been shown to improve whole-body net protein balance to a greater extent than ingesting the same beef in the form of an intrinsically labelled steak^{(Reference Pennings, Groen and van Dijk31)}. Carbohydrate co-ingestion has been shown to further reduce whole-body protein breakdown and amino acid oxidation^{(Reference Gorissen, Burd and Hamer32)}. This may in part be attributed to its insulinotropic properties^{(Reference Groen, Horstman and Hamer37)}.

Apart from nutritional factors, physiological factors also modulate the effect of protein ingestion on whole-body protein metabolism. Resistance- and endurance-type exercises appear to have little-to-no impact on whole-body net protein balance^{(Reference Mazzulla, Parel and Beals40,Reference Trommelen, Holwerda and Kouw42,Reference Holwerda, Kouw and Trommelen43)} . The post-prandial reduction in whole-body protein breakdown rates is attenuated in older adults when compared to younger adults^{(Reference Gorissen, Burd and Hamer32)}. In line, older adults display less of a post-prandial increase in whole-body net balance when compared to younger adults^{(Reference Groen, Horstman and Hamer37)}. Finally, obese individuals seem to display less of a post-prandial increase in whole-body net protein balance when compared to lean individuals following ingestion of 25 g protein. The latter may be explained by the lower dosage when expressed per kg body mass, resulting in less of an increase in post-prandial amino acid availability expressed per kg body mass^{(Reference Kouw, van Dijk and Horstman38)}. Although several factors have been shown to impact whole-body net protein balance, post-prandial changes in plasma amino acid availability seem to represent the main determinant defining the impact of feeding on the post-prandial whole-body net protein balance.

The use of intrinsically labelled protein to quantify post-prandial muscle protein synthesis

Measurement of the muscle protein synthetic response following food (protein) intake is of great importance, but accurate quantification can be challenging. An accurate assessment of muscle protein synthesis requires a steady state in the stable isotope-labelled amino acid precursor pool (i.e. in the plasma or muscle-free amino acid pools). However, protein ingestion is followed by the release of unlabelled amino acids into the circulation, which destabilises the precursor pool(s) by rapid dilution followed by a gradual return to steady-state conditions (Fig. 2a)^{(Reference Burd, Cermak and Kouw50)}. Destabilisation of the precursor pools during the post-prandial period may lower the capacity to detect small changes in muscle protein synthesis rates following food intake (Fig. 2b)^{(Reference Burd, Cermak and Kouw50)}. However, such a destabilisation in the precursor pools can be prevented by the application of intrinsically labelled protein, in which the labelled amino acid in the protein matches the labelled amino acid that is intravenously infused (Fig. 2c). To avoid precursor destabilisation, the labelled amino acid enrichment in the dietary protein must be comparable to the plasma enrichment that will be reached during intravenous-labelled amino acid infusion. Upon protein ingestion, the dietary protein-derived labelled amino acids are digested and absorbed identically to the unlabelled protein-derived amino acids. As a result, the labelled and unlabelled amino acids are uniformly delivered into the plasma and peripheral tissues, which allows maintenance of a steady-state isotope tracer enrichment of the precursor pool(s). This allows for a more sensitive assessment of the muscle protein synthetic response to feeding (Fig. 2d). Although the use of intrinsically labelled protein forms the preferred approach to avoid precursor pool destabilisation during the measurement of post-prandial muscle protein synthesis rates, the co-ingestion of labelled free amino acids with dietary protein represents a less costly alternative. However, free amino acids are much more rapidly absorbed in comparison with intact protein. Therefore, free amino acid co-ingestion may result in a transient increase in precursor enrichment during the early post-prandial period, before eventually returning to steady-state conditions (Fig. 3a). When labelled free amino acids are co-ingested, more frequent plasma sampling should be applied to confirm and appropriately characterise the destabilisation of the post-prandial isotope tracer precursor pool enrichment (Fig. 3b).

Fig. 2. Schematic representation of the use of intrinsically labelled dietary protein to accurately quantify post-prandial muscle protein synthesis rates as well as de novo muscle protein synthesis. (a) and (b): When protein is ingested, the steady-state precursor enrichment obtained by intravenous infusion of stable isotope amino acid tracers is diluted. This dilution of the precursor pool enrichment may compromise the ability to quantify the muscle protein synthetic response to feeding. (c) and (d): When ingesting intrinsically labelled protein, in which the labelled amino acid in the protein matches the labelled amino acid that is intravenously infused, dilution of the precursor pool can be prevented and tracer steady-state may be maintained. This allows for a more accurate measurement of the muscle protein synthetic response to feeding. (e) and (f): The presence of a labelled amino acid (that is not infused) in the dietary protein allows for the assessment of the incorporation of dietary protein-derived amino acids into muscle tissue protein (i.e. de novo muscle protein synthesis).

Fig. 3. Schematic representation of the co-ingestion of labelled free amino acids (corresponding to intravenously infused labelled amino acids) with dietary protein as a means to maintain precursor pool enrichments for the accurate measurement of post-prandial muscle protein synthesis rates. (a): The red dotted line represents dilution of the (infused) labelled amino acid precursor pool following the ingestion of dietary protein. Co-ingestion of the same labelled amino acid allows maintenance of isotope tracer steady-state conditions. (b): Ingestion of a free, crystalline (isotope-labelled) amino acid will be more rapidly absorbed when compared to the ingestion of the same (isotope-labelled) amino acid when it is incorporated into an intact protein. Therefore, co-ingestion of a labelled amino acid cannot prevent disruption of the isotope tracer steady-state. Therefore, it is at all times important to correctly assess changes in precursor pool enrichments over time, which requires a high blood or tissue sampling frequency.

Factors affecting the incorporation of dietary protein-derived amino acids into muscle protein

The use of highly enriched intrinsically labelled protein has consistently shown that dietary protein-derived amino acids are utilised for de novo muscle protein synthesis across a wide variety of populations and can be modulated by various dietary and physiological conditions^{(Reference Groen, Horstman and Hamer3,Reference Kouw, Holwerda and Trommelen27,Reference Trommelen, Kouw and Holwerda51,Reference Fuchs, Kouw and Churchward-Venne52)} . Ingested protein delivers amino acids as precursors for de novo muscle protein synthesis in mixed muscle protein^{(Reference Groen, Horstman and Hamer3)}, and has also been detected in various isolated muscle protein fractions, such as myofibrillar^{(Reference Trommelen, Kouw and Holwerda51)}, mitochondrial^{(Reference Churchward-Venne, Pinckaers and Smeets6)} and connective tissue^{(Reference Trommelen, Holwerda and Senden53)} proteins. Our research group has applied intrinsically labelled protein to demonstrate that the ingestion of 20 g micellar casein protein results in the incorporation of approximately 2 g (about 10 %) dietary protein-derived amino acids into muscle tissue protein during a 5 h post-prandial period^{(Reference Groen, Horstman and Hamer3)}. The observation that dietary protein-derived amino acids are directly incorporated into skeletal muscle protein within the hours following meal ingestion provided us with the evidence that ‘you are what you just ate’^{(Reference Groen, Horstman and Hamer3)}. More recent study has demonstrated that the ingestion of greater amounts of dietary protein results in greater amounts of dietary protein-derived amino acids being incorporated into skeletal muscle protein, with no indication of an upper limit up to the ingestion of a dose of 45 g protein^{(Reference Churchward-Venne, Pinckaers and Smeets6,Reference Holwerda, Paulussen and Overkamp25,Reference Kouw, Holwerda and Trommelen27)} . However, aside from the amount of ingested protein, the type of ingested protein has also been shown to impact dietary protein-derived amino acid incorporation into skeletal muscle protein. In particular, whey protein has been shown to result in a greater incorporation of dietary protein-derived amino acid into skeletal muscle proteins when compared to micellar casein and hydrolysed micellar casein^{(Reference Pennings, Boirie and Senden8)}. This may be explained by the more rapid release of protein-derived amino acids following whey v. casein ingestion and the higher leucine content of whey protein^{(Reference Wall, Hamer and de Lange54,Reference Holwerda, Paulussen and Overkamp55)} .

Besides dietary factors, physiological factors have also been shown to impact the incorporation of dietary protein-derived amino acids into muscle tissue. For instance, prior exercise and neuromuscular electrical stimulation have been shown to result in greater de novo muscle protein synthesis^{(Reference Trommelen, Holwerda and Kouw42,Reference Holwerda, Kouw and Trommelen43,Reference Wall, Burd and Franssen56,Reference Pennings, Koopman and Beelen57)} . Resistance-type exercise enhances the capacity to incorporate dietary protein-derived amino acids into muscle tissue for at least 12 h^{(Reference Wall, Burd and Franssen56)}. However, post-exercise cooling reduces the incorporation of dietary protein-derived amino acids following post-exercise meal intake^{(Reference Fuchs, Kouw and Churchward-Venne52)}. Lastly, short-term muscle disuse results in lower de novo muscle protein synthesis^{(Reference Wall, Dirks and Snijders58)} (Fig. 4). Therefore, it is evident that physical (in)activity is an important factor modulating the capacity to utilise dietary protein-derived amino acids as precursors for de novo muscle protein synthesis.

Fig. 4. Schematic representation of the impact of physical (in)activity on the incorporation of dietary protein-derived amino acids into skeletal muscle protein.