Background
Dietary whey protein has numerous well-known health benefits. For instance, whey protein feeding has been shown to acutely increase postprandial muscle protein synthesis (MPS) in rodents [
1,
2] and humans [
3,
4], whereas chronic whey protein supplementation has been shown to consistently increase muscle mass with exercise training [
5-
7]. Acute whey protein feeding has also been shown to reduce appetite 90–180 min following low-dose ingestion [
8-
10] by potentially affecting anorectic hormone and hypothalamic mRNA expression patterns [
11,
8,
9]. Chronic whey protein supplementation has also been shown to reduce adiposity in rodents and humans [
12-
14,
5]; an effect which may be explained by an increased expression of adipose tissue lipolysis-related gene expression patterns following chronic supplementation [
12], an increase in protein-induced thermogenesis (reviewed in [
15]), and/or a consistent reduction in food intake given its satiety-stimulatory effects as discussed above.
More recent data has focused on the potential health benefits of hydrolyzed dietary proteins. In short, commercial hydrolysis of different dietary protein sources is thought to [
16-
18]: a) expedite the digestion of amino acids via ‘pre-digestion’ thus increasing their postprandial bioavailability; and b) liberate bioactive peptides that are able to exhibit physiological responses that otherwise would be diminished from consuming intact protein sources. Indeed,
in vivo [
19,
20] and
in vitro [
21] evidence suggests that hydrolyzed whey or native whey protein increases the activation of postprandial intramuscular insulin signaling markers. Putative bioactive peptides from whey protein hydrolysates (WPH) have also been shown to exhibit insulin secretagogue properties versus intact whey protein [
22,
23]. Likewise, we have recently demonstrated that acute WPH feeding in rats increases the appearance of di- and oligopeptides as well as numerous lipolysis-related serum markers (i.e., epinephrine, glycerol and numerous free fatty acids) compared to an isonitrogenous WPC feeding condition [
18]. Thus, it is of interest to further examine how WPH versus WPC ingestion differentially affects various physiological systems.
Widespread interest has also surrounded the positive health benefits of dietary egg protein due to its high essential amino acid (EAA) content and high digestibility [
24]. Similar to whey protein, egg protein feeding in rats has been found to significantly increase postprandial MPS [
1]. Likewise, one report suggests that bioactives isolated from egg protein down-regulate serum myostatin (MSTN) [
25]; an effect which may enhance skeletal muscle hypertrophy with chronic supplementation. However, unlike the aforementioned whey protein research, there is a paucity of data regarding the physiological effects of dietary egg protein on other tissues (i.e., adipose tissue and the hypothalamus), though there is some evidence to suggest that egg-based breakfast meals can increase satiety post-ingestion [
26] and cause weight loss in overweight individuals over the long-term [
27].
Given the widespread interest regarding the physiological effects of dietary whey and egg proteins, as well as hydrolyzed versus intact protein forms, the purpose of this study was to examine how different solutions of extensively hydrolyzed whey and egg albumin protein (EPH) blends, in combination with a standardized blend of cow colostrum and egg yolk extract acutely affect post-prandial markers of skeletal muscle anabolism, adipose tissue lipolysis and thermogenesis, and hypothalamic mRNA expression patterns in rodents. Treatments included: 300 human equivalent mg of bovine colostrum and egg yolk extract (0.0057 g protein rat dose) in addition to 10 human equivalent g protein dose (0.19 g protein rat dose) of, a) high-dose WPH + low-dose EPH (70 W/30E); b) equal doses of both WPH and EPH (50 W/50E); and c) low-dose WPH + high-dose EPH (30 W/70E). An isonitrogenous amount of intact whey protein concentrate (WPC) was also fed to a fourth group of rats as a positive feeding control, and 1 ml of water with no protein was fed to a fifth group of rats as a fasting control (CTL). Based upon the aforementioned literature, we hypothesized that all protein treatments would similarly increase postprandial markers of skeletal muscle anabolism as well as satiety-related hypothalamic markers relative to CTL. We also hypothesized that higher proportions of whey protein (i.e., WPC and 70 W/30E) would induce larger increases in adipose tissue lipolysis markers relative to other feeding groups; though we also hypothesized that the hydrolysates would outperform the WPC on markers of muscle anabolism, adipose tissue lipolysis and satiety.
Experimental methods
Animals and feeding protocols
All experimental procedures described herein were approved by Auburn University’s Institutional Animal Care and Use Committee. Male Wistar rats (~250 g) approximately 8–9 weeks old were purchased from Harlan Laboratories and were allowed to acclimate in the animal quarters for 5 days prior to experimentation. Briefly, animal quarters were maintained on a 12 h light: 12 h dark cycle, at ambient room temperature, with water and standard rodent chow (18.6% protein, 44.2% carbohydrate, 6.2% fat; Teklad Global #2018 Diet, Harlan Laboratories) provided to animals ad libitum.
The day prior to acute protein feeding experiments, food was removed from home cages resulting in an 18 h overnight fast. The morning of experimentation, animals were removed from their quarters between 0800–0900, transported to the Molecular and Applied Sciences Laboratory and were allowed to acclimate for approximately 3–5 h. Thereafter, rats were administered either WPC, 70 W/30E, 50 W/50E, 30 W/70E at a human equivalent (eq.) dose of 10 g protein (0.19 g protein rat dose) dissolved in 1 ml of tap water via gavage feeding. Doses were calculated per the species conversion calculations of Reagan-Shaw et al. [
28], whereby the human body mass for an average male was assumed to be 80 kg. The group of non-fed CTL rats was gavage-fed 1 ml of tap water. Dietary components of each test protein solution are presented in Table
1.
Table 1
Contents of each protein per the 0.19 g protein dose of each respective protein
Alanine | 10 | 10 | 10 | 9 |
Arginine | 8 | 8 | 9 | 6 |
Aspartic Acid | 21 | 20 | 19 | 22 |
Cysteine | 5 | 5 | 5 | 4 |
Glutamic Acid | 31 | 29 | 26 | 34 |
Glycine | 4 | 5 | 5 | 4 |
Histidine* | 4 | 4 | 4 | 4 |
Isoleucine*† | 11 | 11 | 10 | 12 |
Leucine*† | 20 | 19 | 17 | 23 |
Lysine* | 17 | 15 | 14 | 19 |
Methionine* | 5 | 5 | 6 | 4 |
Phenylalanine* | 8 | 9 | 9 | 7 |
Proline | 12 | 11 | 9 | 17 |
Serine | 12 | 12 | 11 | 12 |
Threonine* | 11 | 10 | 9 | 12 |
Tryptophan* | 3 | 3 | 3 | 3 |
Tyrosine | 7 | 7 | 7 | 6 |
Valine*† | 12 | 12 | 12 | 11 |
Total EAAs*
|
92
|
87
|
83
|
95
|
Total BCAAs*†
|
43
|
41
|
38
|
46
|
M.W.
|
70 W/30E
|
50 W/50E
|
30 W/70E
|
WPC
|
(kDa)
|
(%)
|
(%)
|
(%)
|
(%)
|
<1.0 | 40 | 39 | 40 | 0 |
1.0 - 5.0 | 23 | 22 | 23 | 7 |
5.0 - 10.0 | 6 | 6 | 6 | 11 |
>10.0 | 30 | 32 | 32 | 82 |
Of note, We examined how graded doses of WPC in solution (0.19, 0.37, and 0.93 g protein) stimulated MPS and Akt-mTOR markers 90 min post-gavage in order to determine an optimal dose that adequately elicited a post-prandial physiological response. These preliminary results demonstrated that 10 human eq. g of WPC (0.19 g protein) increased markers of mTOR activation and MPS 90 min post-gavage, and this generally was equal to the 19 human eq. g (0.37 g protein) and 48 human eq. g (0.93 g protein) doses (Additional file
1: Figure S1). Thus, given that the 10 human eq. g of WPC (0.19 g protein) elicited similar anabolic responses compared to higher doses, we opted to use the 10 human eq. g (0.19 g) dose for each test protein. While this dose is not typically associated with the optimal human MPS response to protein ingestion (i.e., 20–40 g), it should be noted that the species conversion calculations of Reagan-Shaw et al. is a basis to dose rats relative to humans and, alternatively, these human eq. dosages should not be viewed in absolute terms when comparing species (i.e., 10 human eq. g appears to elicit an anabolic response in rats whereas 20–40 g in humans is needed).
The gavage feeding procedure involved placing the animals under light isoflurane anesthesia for approximately 1 min while gavage feeding occurred. Following gavage feeding, rats were allowed to recover 90 or 180 min prior to being euthanized under CO
2 gas in a 2 L induction chamber (VetEquip, Inc., Pleasanton, CA, USA). Animals that were sacrificed 180 min post-treatment were injected intraperitoneally with puromycin dihydrochloride (5.44 mg in 1 ml of diluted in phosphate buffered saline; Ameresco, Solon, OH, USA) 30 min prior to euthanasia in order to determine skeletal muscle protein synthesis via the surface sensing of translation (SUnSET) method described in detail elsewhere [
29]. Of note, with the SUnSET method MPS is determined through the incorporation of puromycin into actively synthesized proteins given that it is a structural analogue of aminoacyl-transfer RNA; specifically tyrosyl-tRNA. It should also be noted that the SUnSET method is an alternative method for measuring MPS compared to radioactive isotope (e.g.
3H-phenyalanine or
35S-methionine), or stable isotope (e.g.
15 N-lysine,
13C-leucine or [ring-
13C6]-phenylalanine) tracers. Goodman et al. [
29] compared the SUnSET method to a
3H-phenyalanine flooding method in
ex vivo plantaris muscle preparations isolated from animals that had undergone synergist ablation. Remarkably, these authors determined that MPS rates increased 3.6-fold as determined by the SUnSET method and 3.4-fold as determined by the tracer method; a finding which proves the reliability of this method in detecting sensitive changes in MPS.
Immediately following euthanasia, whole blood was removed via heart sticks using a 21-gauge needle and syringe, placed in a serum separator tubes, and processed for serum extraction via centrifugation at 3,500 ×
g for 5 min. Serum was aliquoted into multiple 1.7 ml microcentrifuge tubes for subsequent biochemical assays and then frozen for later analysis. Approximately two 50 mg pieces of mixed gastrocnemius muscle was harvested using standard dissection techniques and placed in homogenizing buffer [Tris base; pH 8.0, NaCl, NP-40, sodium deoxycholate, SDS with added protease and phosphatase inhibitors (G Biosciences, St. Louis, MO, USA)] and Ribozol (Ameresco) for immunoblotting and mRNA analyses, respectively. Approximately two 50 mg pieces of subcutaneous adipose tissue (SQ) from the inguinal crease was harvested and placed in the aforementioned Tris base homogenizing buffer and Ribozol for immunoblotting and mRNA analyses, respectively. Due to tissue limitations, only one 50 mg piece of omental adipose tissue (OMAT) was harvested and placed in the aforementioned Tris base homogenizing buffer for immunoblotting. Finally, removal of the hypothalamus was performed per the methods similar to those previously employed [
30]. Briefly, brains were removed and rinsed in 1x phosphate buffered saline. Brains were then placed posterior side up in a 1.0 mm acrylic sectioning apparatus (Braintree Scientific, Braintree, MA, USA) and a 2.0-mm coronal slice of each brain was made between Bregma-1.6 and-1.8 mm. Coronal slices were immediately placed on an ice-cooled stage and two bilateral punches (2.0 mm diameter) were made to capture the hypothalamus. Tissue was immediately placed in Ribozol and stored at-80°C until RNA isolation.
Gastrocnemius muscle, SQ and OMAT samples placed in Tris base homogenizing buffer were homogenized using a 1.7 ml tube using a tight-fitting micropestle, insoluble proteins were removed with centrifugation at 500 × g for 5 min at 4°C, and supernatants were assayed for total protein content using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) prior to immunoblotting sample preparation. Muscle, SQ, and hypothalamus samples placed in Ribozol were subjected to total RNA isolation according to manufacturer’s instructions, and concentrations were performed using a NanoDrop Lite (Thermo Scientific) prior to cDNA synthesis for mRNA analyses. Extra gastrocnemius muscle and SQ fat not processed during dissections were flash-frozen in liquid nitrogen and stored at-80°C for later potential analyses.
Directed Akt-mTOR phosphoproteomics
The PathScan® Akt Signaling Antibody Array Kit (Chemiluminescent Readout; Cell Signaling, Danvers, MA, USA) containing glass slides spotted with antibodies was utilized to detect phosphorylated proteins predominantly belonging to the Akt-mTOR signaling network.
The kit assays p-Akt (Thr308), p-Akt (Ser473), p-rps6 (Ser235/236), p-AMPKα (Thr172), p-Pras40 (Thr246), p-mTOR (Ser2481), p-GSK-3α (Ser21), p-GSK-3β (Ser9), p-p70s6k (Thr389), p-p70s6k (Thr421/Ser424), p-BAD (Ser112), p-PTEN (Ser380), p-PDK1 (Ser241), p-ERK1/2 (Thr202/Tyr204), p-4E-BP1 (Thr37/46). However, we specifically analyzed p-Akt (Ser473), p-rps6 (Ser235/236), p-AMPKα (Thr172), p-mTOR (Ser2481), p- p-p70s6k (Thr389), and p-4E-BP1 (Thr37/46) in order follow a ‘linear’ analysis in Akt-mTOR signaling. Briefly, gastrocnemius homogenates were diluted to 0.5 μg/μl using cell lysis buffer provided by the kit and assayed according to manufacturer’s instructions. Slides were developed using an enhanced chemiluminescent reagent provided by the kit, and spot densitometry was performed through the use of a UVP Imager and associated densitometry software (UVP, LLC, Upland, CA, USA). The calculation of each phosphorylated target was as follows:
(Density value of the target – negative control)/summation of all density values for the sample.
It should be noted that this high throughput antibody chip array for muscle phosphorylation markers was used rather than single antibodies due to resource constraints. Notwithstanding, and as discussed in the results section, the results presented herein are in agreement with past literature showing that protein feeding affects numerous targets on the aforementioned antibody array chip. Furthermore, our preliminary WPC graded-dose feedings show an increase in Akt-mTOR markers across multiple doses relative to fasting rats (Additional file
1: Figure S1). We have also internally tested this array on exercised rat muscle as well as C2C12 cell culture lysates deprived of or treated with L-leucine, and have produced reproducible results commensurate with prior literature examining these markers (i.e., increased activation of mTOR markers which parallel increases in MPS;
unpublished observations).
Western blotting
As mentioned prior, the SUnSET method was employed in order to examine if different dietary protein blends differentially affected MPS. Briefly, 2 μg/μl gastrocnemius Western blotting preps were made using 4x Laemmli buffer. Thereafter, 20 μl of prepped samples were loaded onto pre-casted 4–20% SDS-polyacrylamide gels (C.B.S. Scientific Company, San Diego, CA, USA) and subjected to electrophoresis (200 V @ 75 min) using pre-made 1x SDS-PAGE running buffer (C.B.S. Scientific Company). Proteins were then transferred to polyvinylidene difluoride membranes, and membranes were blocked for 1 h at room temperature with 5% nonfat milk powder. For muscle samples, mouse anti-puromycin IgG (1:5,000; Millipore) was incubated with membranes overnight at 4°C in 5% bovine serum albumin (BSA), and the following day membranes were incubated with anti-mouse IgG secondary antibodies (1:2,000, Cell Signaling) at room temperature for 1 h prior to membrane development described below. Thereafter, membranes were stripped of antibodies via commercial stripping buffer (Restore Western Blot Stripping Buffer, Thermo Scientific), membranes were incubated with rabbit anti-beta-actin (1:5,000; GeneTex, Inc., Irvine, CA, USA) as a normalizer protein overnight at 4°C in 5% BSA, and the following day membranes were incubated with anti-rabbit IgG secondary antibodies (1:2,000, Cell Signaling) at room temperature for 1 h prior to membrane development.
SQ and OMAT samples were assayed with rabbit anti-phospho-hormone sensitive lipase [p-HSL (Ser563) IgG (1:1000; Cell Signaling)] overnight at 4°C in 5% BSA. The following day membranes were incubated with anti-rabbit IgG secondary antibodies (1:2,000, Cell Signaling) at room temperature for 1 h prior to membrane development. Membranes were stripped, incubated with rabbit glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000; GeneTex) overnight at 4°C in 5% BSA, and the following day were incubated with anti-rabbit IgG secondary antibodies (1:2,000, Cell Signaling) at room temperature for 1 h prior to membrane development.
Membrane development was performed using an enhanced chemiluminescent reagent (Amersham, Pittsburgh, PA, USA), and band densitometry was performed through the use of a UVP Imager and associated densitometry software (UVP, LLC, Upland, CA, USA).
Real-time RT-PCR
RNA from each tissue (500 ng of hypothalamus RNA and 1 μg of gastrocnemius and SQ RNA) were reverse transcribed into cDNA for real time PCR analyses using a commercial cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD, USA). Real-time PCR was performed using SYBR-green-based methods with gene-specific primers [MSTN, Mighty/Akirin-1, Myosin Heavy Chain 4 (Myhc4), p21Cip1, Atrogin-1, MuRF-1, GLUT-4, Insulin-like growth factor-1ea (IGF-1Ea), proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related protein (AGRP), leptin receptor (LEPR), peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), uncoupling protein 3 (UCP3), carnitine palmitoyltransferase 1b (CPT1B), beta-2 microglobulin (B2M), and beta-actin] designed using primer designer software (Primer3Plus, Cambridge, MA, USA). The forward and reverse primer sequences are as follows: [MSTN: forward primer 5′-ACGCTACCACGGAAACAATC-3′, reverse primer 5′-CCGTCTTTCATGGGTTTGAT-3′; Mighty/Akirin-1: forward primer 5′-TTTGATCTTGGGGATTCTGG-3′, reverse primer 5′-GCCTGGAAACAGTCCCTGTA-3′; p21Cip1: forward primer 5′-AGCAAAGTATGCCGTCGTCT-3′, reverse primer 5′-ACACGCTCCCAGACGTAGTT-3′; Atrogin-1: forward primer 5′-CTACGATGTTGCAGCCAAGA −3′, reverse primer 5′- GGCAGTCGAGAAGTCCAGTC-3′; MuRF-1: forward primer 5′-AGTCGCAGTTTCGAAGCAAT-3′, reverse primer 5′-AACGACCTCCAGACATGGAC-3′; GLUT-4: forward primer 5′-GCTTCTGTTGCCCTTCTGTC-3′, reverse primer 5′-TGGACGCTCTCTTTCCAACT-3′; IGF-1Ea: forward primer 5′-TGGTGGACGCTCTTCAGTTC-3′, reverse primer 5′-TCCGGAAGCAACACTCATCC-3′; POMC: forward primer 5′-GAAGGTGTACCCCAATGTCG-3′, reverse primer 5′-CTTCTCGGAGGTCATGAAGC-3′; NPY: forward primer 5′-AGAGATCCAGCCCTGAGACA-3′, reverse primer 5′-AACGACAACAAGGGAAATGG-3′; AGRP: forward primer 5′-CGTGTGGGCCCTTTATTAGA-3′, reverse primer 5′-CAGACCTTCTGATGCCCTTC-3′; LEPR: forward primer 5′-CTGGGTTTGCGTATGGAAGT-3′, reverse primer 5′-CCAGTCTCTTGCTCCTCACC-3′; PGC-1α: forward primer 5′-ATGTGTCGCCTTCTTGCTCT-3′, reverse primer 5′-ATCTACTGCCTGGGGACCTT-3′; UCP3: forward primer 5′-GAGTCAGGGGACTGTGGAAA-3′, reverse primer 5′-GCGTTCATGTATCGGGTCTT-3′; CPT1B: forward primer 5′-CCCAGTTCTGAGACCAGCTC-3′, reverse primer 5′-TAGGCACCTAAGGGCTGAGA-3′; B2M: forward primer 5′-CCCAAAGAGACAGTGGGTGT-3′, reverse primer 5′-CCCTACTCCCCTCAGTTTCC-3′; beta-actin: forward primer 5′-GTGGATCAGCAAGCAGGAGT-3′, reverse primer 5′-ACGCAGCTCAGTAACAGTCC-3′] and SYBR green chemistry (Quanta). Primer efficiency curves for all genes were generated and efficiencies ranged between 90% and 110%, and melt curve analyses demonstrated that one PCR product was amplified per reaction.
SQ cAMP determination
Frozen SQ samples were subjected to 3'–5'-cyclic adenosine monophosphate (cAMP) assays using a rat-specific spectrophotometric commercial assay (R&D Systems, Inc., Minneapolis, MN, USA). Briefly, approximately 50–100 mg of tissue was homogenized in 500 μl of 0.1 N HCl. Samples were subjected to 10 min of centrifugation at 10,000 × g at 4°C, and neutralized with 50 μl of 1 N NaOH. Samples were then diluted 2-fold with the assay diluent provided, and cAMP concentrations were determined according to the manufacturer’s recommendations.
Serum analyses
Serum samples were assayed for lipolysis markers including free fatty acids (FFAs) as well as epinephrine (EPI) and norepinephrine (NorEPI) using rat-specific spectrophotometric commercial assays according to the manufacturer’s recommendations (FFAs: Abcam, Cambridge, MA, USA; EPI/NorEPI: Abnova, Taipei City, Taiwan). Serum samples were also analyzed for triiodothyronine (T3) using a rat-specific spectrophotometric commercial assay according to the manufacturer’s recommendations (Abnova).
Statistics
All data are presented in figures and tables as means ± standard error values. Given that each post-treatment time point were comprised of independent groups of rats, statistical comparisons were performed using one-way ANOVAs, and statistical significance was set at p < 0.05 (SPSS v 22.0, IBM, Armonk, NY, USA). When between-group significance was obtained, a Fisher’s LSD post hoc test was performed in order to determine specific between-group comparisons.
Conclusions
We have demonstrated that protein type provide uniquely different physiological responses over a transient post-prandial time course. Specifically, and seemingly irrespective of protein type, administering higher concentrations of whey versus egg protein to healthy rodents causes: a) a greater anabolic response in rodents with regards to post-feeding MPS compared to a fasting condition; and b) an increase in intramuscular insulin sensitivity markers (i.e., Akt signaling markers and transient increases in PGC-1α mRNA expression patterns). Alternatively, the administration of higher concentrations of WPH versus EPH increases select markers of post-feeding lipolysis 3 h post-feeding. Of note, while we make assertions that whey protein forms may be more beneficial in facilitating increases in muscle mass and fat loss compared to egg protein per the current findings, the acute nature of this study is a pervading limitation of these hypotheses. Likewise, while several of tissue markers were statistically altered in response to different protein feedings, more research is needed comparing whey versus egg protein supplementation on longer-term physiologically-relevant outcomes (i.e., increases in muscle mass, decreases in fat mass, and/or alterations in satiety as suggested by our transient findings reported herein). Therefore, further research is this nutraceutical arena is warranted with regards to how protein source and type (i.e., native versus hydrolyzed), and varying combinations thereof may affect these physiological parameters in over more chronic periods and in more clinical-based populations.
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Competing interests
Besides C.M.L., none of the authors have non-finacial and/or financial competing interests. C.M.L. is employed by 4Life, but he intellectually contributed to study design and data write-up. Therefore, all co-authors agreed that his intellectual input into this project warranted co-authorship.
Authors’ contributions
CBM, CDF, BSF, CAP, JCH, JSM, CML and MDR: This person has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. CBM, CML and MDR: This person primarily was involved in drafting the manuscript or revising it critically for important intellectual content. CBM, CDF, BSF, CAP, JCH, JSM, CML and MDR: This person gave final approval of the version to be published. CBM, CDF, BSF, CAP, JCH, JSM, CML and MDR: This person agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.