Protein type is an important factor in acutely increasing markers of skeletal muscle anabolism

Whey and egg protein consumption has been posited to promote anabolic effects in skeletal muscle via greater post-feeding increases in serum amino acids versus other protein sources [2]. All test proteins in the current study increased the phosphorylation status of mTOR, p70s6k, and rps6 90 min post-feeding compared to CTL rats, though 70 W/30E-fed rats presented sustained elevations in phosphorylated mTOR and rps6 180 min post-feeding. These phosphorylated targets are positive effectors of MPS, and our findings are in agreement with past literature suggesting that whey and egg protein increase the phosphorylation of one or more of these intramuscular signaling markers following feeding with [19,34,20,35] or without [2,1] resistance exercise in rats and humans. However, it is intriguing that higher proportions of EPH (i.e., 50–70%) did not statistically increase MPS levels versus CTL rats. Norton et al. [1] demonstrated that a test meal containing 0.64 g of whey or egg protein similarly increases MPS 90 min post-feeding. Our study differs from the findings of Norton et al. given that: a) MPS was measured using two different methodologies; specifically we used the SUnSET method and Norton et al. used an L-2 H 5 -phenylalanine tracer; b) Norton et al. measured post-feeding MPS at 90 min while we measured MPS 180 min post-feeding; and c) Norton et al. fed rats 0.64 g protein in a solid mixed-meal form while we fed rats 0.19 g of unadulterated test protein solutions. In spite of these methodological differences, we suggest that, relative to CTL rats, a low protein dose comprised mainly of whey protein (i.e., WPC or 70 W/30E) promotes a greater post-feeding increase in MPS relative to a low dose protein solution comprised primarily of egg protein. Alternatively stated, while egg protein is a source of leucine and EAAs, it appears that whey protein is superior at stimulating MPS at lower doses in the current rodent model. While this seems contrary to the conclusions posited by Norton et al. suggesting that the high leucine content in whey and egg equally stimulate MPS, two independent human studies have demonstrated that younger [36] and older subjects [37] consuming supplemental egg protein while resistance training do not experience increases in muscle mass after 8–12-week interventions. Specifically, Hida et al. [36] demonstrated that 15 g/d of egg protein supplementation in female athletes, who were engaged in a resistance training protocol, increased lean body mass by 1.5 kg, whereas a carbohydrate placebo increased lean body mass by 1.6 kg. Likewise, Iglay et al. [37] demonstrated that supplementing the diet with an additional 20 g/d of egg protein did not further increase the lean mass or skeletal muscle cross-sectional area compared to a lower protein group when both groups resistance trained for 12 weeks; of note, both groups gained roughly 1.0 kg of lean body mass.

In contrast, a recent meta-analysis examining several studies [5] clearly demonstrates that whey protein supplementation with resistance exercise is effective at increasing muscle mass in younger and older populations, and Phillips et al. [6] noted that participants engaged in 8–16 weeks of resistance exercise gain, on average, 3.0 kg of lean mass compared to 1.0 kg of lean mass gains in the placebo groups of these studies. One hypothesis deserving of future investigation is whether mammary-derived proteins, due to the inherent purpose of such proteins promoting rapid growth and development of offspring, may offer unique physiological advantages versus what can otherwise be labeled as ‘nutritional protein sources’ such as egg or other animal proteins. In this regard, future studies examining why a low dose of whey protein is unique in stimulating muscle anabolism relative to other protein sources that possess a ‘leucine-, BCAA-, and EAA-rich profile’ are warranted.

Putative anabolic and atrogene gastrocnemius mRNA responses following different protein feedings

Akirin-1/Mighty increased approximately 90% 180 min post-feeding in the WPC and 70 W/30E groups versus CTL rats and other protein groups. Akirin-1/Mighty is a transcriptional target of MSTN that is related to controlling myotube size in vitro [32], and resistance exercise has been shown to transiently up-regulate Akirin-1/Mighty mRNA in rodent skeletal muscle [31]. To our knowledge, only one other recent study to date has determined that certain akirin genes are transcriptionally up-regulated in fish that were fasted 21 days and then re-fed [38]. Hence, the aforementioned study along with our current data suggests that Akirin-1/Mighty mRNA is sensitive to protein feeding, and this finding should be further examined at the mechanistic level in order to determine if whey protein affects skeletal muscle hypertrophy through increases in Akirin-1/Mighty mRNA expression.

The expression of select anabolic and catabolic-related gastrocnemius mRNAs responded differently between different treatment groups. Interestingly, higher proportions of EPH caused 90–180 min increases in MSTN mRNA versus CTL rats and/or higher proportions of whey protein. Preliminary data in humans suggest that the consumption of fertile egg yolk powder reduces circulating MSTN levels [25]. Hence, if one or multiple putative bioactive components in egg protein extract reduce serum MSTN levels then it is possible that skeletal muscle may undergo a compensatory increase in skeletal MSTN mRNA expression to counter systemic down-regulation. Thus, while our data and other limited evidence suggests that MSTN expression is responsive to dietary egg proteins, more research is needed in order to elucidate if egg protein-induced increases in MSTN gene expression and/or signaling in skeletal muscle results in a physiological meaningful response.

All protein sources generally increased the p21Cip1 mRNA expression 180 min post-feeding compared to CTL rats suggesting that protein feeding in general regulates the expression of this gene. p21Cip1 gene expression has been theorized to promote satellite cell differentiation [39,40], though limited information suggests that p21Cip1 gene expression up-regulates protein synthesis and pathological hypertrophy in kidney epithelial cells [41]. Thus, it will be of further interest to examine if protein feeding-induced increases in skeletal muscle p21Cip1 gene expression are related to post-mitotic skeletal muscle protein synthesis mechanisms.

Atrogin-1 was up-regulated in 70 W/30E-fed rats 180 min post-feeding versus CTL rats. Similarly, MuRF-1 was up-regulated in WPC-fed and 70 W/30E-fed rats 180 min post-feeding versus CTL rats. Our finding that test solutions containing predominantly whey protein increase postprandial atrogene (atrogin-1 and MuRF-1) mRNA expression is intriguing given that amino acids are thought to be anti-catabolic [42]. However, ingesting smaller protein ingestion boluses (10–20 g) have been reported to increase MuRF-1 mRNA in human skeletal muscle after resistance exercise versus a larger bolus (40 g) [43]. Thus, our finding that protein ingestion increases the mRNA expression of select atrogenes may represent a stimulation of greater muscle protein turnover rather than an increase in atrophic mechanisms.

Protein source and type as important factors in acutely affecting markers of skeletal muscle metabolism and reduced muscle catabolism

Higher proportions of whey protein in the test solutions (i.e., WPC and 70 W/30E) increased Akt phosphorylation (Ser473) 90 min post-feeding versus CTL rats. Tissue Akt phosphorylation at the Ser473 residues is a common readout for insulin signaling and sensitivity [44], and whey protein feeding following resistance exercise in humans has been shown to increase Akt phosphorylation at the Ser473 residue [19,20]. Our findings are also in partial agreement with West et al. [45] who demonstrated in humans that an EAA bolus increases skeletal muscle Akt phosphorylation (Ser473) 60 min after feeding. As noted above, however, WPC and EPH are also a rich source of EAAs. Thus, we speculate that the increase in Akt phosphorylation in the WPC and 70 W/30E groups may have been due to the superior ability of whey protein in stimulating insulin secretion and, thus, downstream insulin signaling in skeletal muscle. While we did not measure serum insulin responses in the current study, we have previously shown that WPH feeding to rats causes a robust (>2-fold) rise in insulin 60 min post-feeding [23]. Hence, foods containing a higher proportion of whey protein may stimulate greater intramuscular insulin signaling, and future research should continue to examine if WPC or WPH feeding in acute and long-term settings can enhance insulin sensitivity in insulin-resistant subjects.

Interestingly, 70 W/30E feeding caused a 63% increase in skeletal muscle PGC-1α mRNA expression versus CTL rats, as well as a significant increase in this gene relative to all other groups 90 min post-treatment. Furthermore, rats fed 70 W/30E exhibited a significant increase in skeletal muscle CPT1B mRNA 90- and 180 min post-feeding; this being a gene which is involved with fatty acid transport to the mitochondria for fuel oxidation. Whey protein isolate has been shown to stimulate a further increase in PGC-1α mRNA expression in human skeletal muscle 6 h following cycling [46]. However, to our knowledge, this is the first study to demonstrate that a test protein containing chiefly WPH can increase post-feeding skeletal muscle PGC-1α mRNA expression independent of exercise. We posit that one potential mechanism whereby WPH stimulates the mRNA expression of PGC-1α and CPT1B is through the stimulation of AMPK activity (Figure 3a). To this end, Canto et al. [47] have demonstrated that AMPK activation increases the expression of these two genes, and this would support the hypothesis that whey protein, in particular WPH, can stimulate oxidative metabolism and mitochondrial biogenesis with long-term supplementation. This hypothesis is not unfounded given recent evidence that prolonged whey protein feeding has been shown to increase mitochondrial content and respiration in the brain [48] and liver [49]. Therefore, more mechanistic studies should examine if WPH administration increases the post-feeding expression of mitochondrial-related genes via AMPK activation and/or other mechanisms.

Effects of different proteins on post-feeding markers of lipolysis

As mentioned prior, whey protein ingestion exerts positive effects on body composition and fat mass [14,5]. Furthermore, and as mentioned previously, WPH supplementation during exercise may provide added benefit to reducing body fat versus intact/native protein sources. Despite a transient 90 min post-feeding depression in serum FFAs with 70 W/30E feeding versus CTL rats and other protein groups, 50–70% WPH protein feedings increased select markers of adipose tissue lipolysis and thermogenesis 180 min post-feeding. For instance, rats fed 70 W/30E presented increases in SQ cAMP levels as well as OMAT and SQ p-HSL (Ser563). Likewise, rats that were fed higher proportions of WPH (e.g., 70 W/30E or 50 W/50E) exhibited increases in SQ PGC-1α and UCP3 mRNA expression levels which are putative markers of adipose tissue thermogenesis [50]. Finally, 70 W/30E increased gastrocnemius CPT1B mRNA which could be suggestive of a potential long-term enhancement in fatty acid transport to the mitochondria for oxidation. Conversely, circulating catecholamine levels in response to feeding higher proportions of WPH exhibited no discernable effects. These findings are difficult to reconcile as we have previously reported that WPH increases serum EPI 30 min post-feeding versus WPC-fed and CTL rats [18]. Therefore, the 180-min post-feeding increase in lipolysis markers in the current study may be due to an earlier increase in catecholamines (i.e., within 60 min of feeding) which was not captured due to sampling time points and/or due to WPH-borne bioactives that selectively act upon adipose tissue to stimulate lipolytic mechanisms.

Of note, we measured serum T3 given that it is a well-known stimulator of thermogenesis and cellular respiration. With regards to adipose tissue lipolysis, T3 has been shown to increase adipocyte beta-adrenergic receptor which, in turn, increases lipolytic capabilities over longer-term periods [51]. Notwithstanding, there was no clear protein feeding effect on serum T3 depression, and T3 values did not seem to parallel the increased lipolysis and thermogenesis markers in rats fed 70 W/30E or 50 W/50E which refutes the potential role of thyroid hormones in facilitating this effect.

One final mechanistic explanation as to how higher proportions of WPH increased lipolysis markers is through potential tricarboxylic acid (TCA) cycle modulation. To this end, a recent study by Lillefosse et al. [52] demonstrated that chronic whey protein feeding to obese-prone rodents significantly reduced fat mass gain in response to concomitant high fat feeding. The authors suggested that whey protein feeding increases the urinary excretion of TCA substrates which are stimulators of fatty acid synthesis [53]. Alternatively stated, the ability of WPH to ‘extract’ TCA cycle intermediates from adipose tissue during the post-feeding period may place adipose tissue in a catabolic state thereby initiating lipolysis-related mechanisms. This is not unfounded, as we have previously noted that WPH significantly increases circulating TCA intermediates (i.e., citrate, succinate, fumarate and malate) 60 min post-feeding versus WPC-fed rats (supplementary data in [18]). Hence, more research is needed regarding if the depletion of TCA cycle intermediates within adipose tissue is linked to the WPH-induced lipolysis response.

Effects of different proteins on post-feeding markers of satiety

Sousa et al. [54] recently posited that, regardless of protein source, amino acids may reduce appetite via an increase in gut hormone secretion, an increase in anorexigenic POMC gene expression in the hypothalamus, and/or a reduction in orexigenic NPY gene expression in the hypothalamus. 70 W/30E and 30 W/70E increased hypothalamic POMC mRNA expression patterns 90 min post-feeding; this being a marker that favors satiety signaling in the hypothalamus [55]. However, there was a compensatory increase in the orexigenic AGRP transcript in rats fed a high proportion of WPH. Furthermore, some protein feedings induced an increased expression of hypothalamic NPY mRNA versus CTL rats which, again, suggests a potential orexigenic versus satiety response. Therefore, our mixed findings suggest that two possibilities may exist including: a) the amount of total protein fed to rats, while beneficial in stimulating skeletal muscle anabolism and adipose tissue lipolysis, was not entirely effective at initiating a satiety response; and/or b) hypothalamic signaling is so tightly regulated that a post-feeding increase in anorectic genes is countered with a compensatory increase in orexigenic genes.

Finally, it should be noted that the post-feeding effects of each protein on hypothalamic LEPR mRNA expression patterns was of considerable interest due to the central role of leptin receptor signaling in satiety. Thus, we initially hypothesized that protein-feeding induced alterations in LEPR mRNA expression may be a potential culprit in initiating longer-term body composition alterations through enhanced satiety mechanisms that have been reported to previously occur with chronic protein supplementation. To this end, McAllan et al. [11] recently performed a long-term rodent feeding study whereby C57BL/6 J mice were fed a high fat diet (HFD, 45% energy as fat) enriched with either 20% energy as casein or whey protein isolate. HFD feeding increased the hypothalamic mRNA expression of LEPR; an effect which the authors suggest may be a hallmark feature of hyperphagia and obesity development. However, mice that were co-fed whey protein isolate with the HFD presented a significant reduction in hypothalamic LEPR mRNA expression. Notwithstanding, we demonstrated no noticeable between-group differences in LEPR mRNA expression patterns which suggests that the hypothalamic expression gene is not appreciably altered after one feeding and/or LEPR gene expression may be indiscriminately regulated more so by amino acid concentration alone as opposed to specific bioactive peptides.