Muscle Protein Signaling in C2C12 Cells Is Stimulated to Similar

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Muscle Protein Signaling in C2C12 Cells is Stimulated to a Similar Degree by Diverse Commercial Food Protein Sources and Experimental Soy Protein Hydrolysates David A Roeseler, Nancy J McGraw, Dustie N Butteiger, Naina Shah, Janine Hall-Porter, Ratna Mukherjea, and Elaine Susan Krul J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05460 • Publication Date (Web): 24 Mar 2017 Downloaded from http://pubs.acs.org on March 29, 2017

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Journal of Agricultural and Food Chemistry

Roeseler DA et al.

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Title: Muscle Protein Signaling in C2C12 Cells is Stimulated to a Similar Degree by

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Diverse Commercial Food Protein Sources and Experimental Soy Protein Hydrolysates

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Authors: David A. Roeseler1, Nancy J. McGraw2, Dustie N. Butteiger3, Naina Shah4,

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Janine Hall-Porter5, Ratna Mukherjea* and Elaine S. Krul6

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Work was conducted at DuPont Nutrition & Health, St. Louis, MO, USA

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*Corresponding author: Ratna Mukherjea, Ph.D. DuPont Nutrition & Health 4300 Duncan Ave St. Louis, MO, 63110 Phone: +1-314-659-3135; FAX: +1-314-659-5733 Email: [email protected] 1

Current address: Patheon Biologics 4766 La Guardia Dr, St. Louis, MO 63134 Email: [email protected] 2

Current address: Eurofins Pharma Bioanalytics Services US Inc. 15 Research Park Drive, St. Charles, MO 63304 Email: [email protected] 3

Current address: Stereotaxis 4320 Forest Park Ave., Suite 100, St. Louis, MO 63108 Email: [email protected] 4

Current address: PepsiCo Global Flavors, Seasonings and Ingredients 7701 Legacy Drive; Mail Stop 3T-125; Plano, TX 75024-4099 Email: [email protected]

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Title running header: Muscle protein signaling by food proteins

Current address: Nestlé-Purina Checkerboard Square, St. Louis, MO 63164 Email: [email protected] 6

Current address: EKSci, LLC 594 Gederson Lane, St. Louis, MO 63122 Email: [email protected]

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Abstract

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Dietary protein stimulates muscle protein synthesis and is essential for muscle health. We

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developed a screening assay using C2C12 mouse muscle cells to assess the relative abilities of

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diverse commercial proteins sources and experimental soy protein hydrolysates (ESH), after

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simulated gut digestion (SGD), to activate the mechanistic target of rapamycin complex I

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(mTORC1) muscle protein synthesis signaling pathway (p70S6K(Thr389) phosphorylation).

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Activation of mTORC1 was expressed as a percentage of a maximal insulin response. The

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bioactivity of proteins grouped by source including: fish (81.3±10.6%), soy (66.2±4.7%), dairy

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(61.8±4.3%), beef (53.7±8.6%), egg (52.3±10.6%), soy whey (43.4±8.6%), and pea

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(31.4±10.6%) were not significantly different from each other. Bioactivity for ESH ranged from

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28.0±7.5% to 98.2±6.6%. The results indicate that both the protein source and processing

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conditions are key determinants for mTORC1 activation. Regression analyses demonstrated

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that neither leucine nor total branched chain amino acid content of proteins are sole predictors

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of mTORC1 activity and that additional factors are necessary.

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Key words: soy, dairy, protein, mTORC1, muscle,

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Introduction

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High-quality dietary proteins are an essential macronutrient for promoting muscle and

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overall metabolic health 1. The muscle health benefits of dietary protein intake are of

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great interest to individuals engaged in sports, on weight loss regimens and those in

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aging populations. The gain in skeletal muscle mass is highly dependent on a net

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increase in muscle protein synthesis (MPS) over muscle protein breakdown (MPB) 2.

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While numerous clinical studies have investigated the beneficial effect of dietary protein

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sources on MPS in healthy individuals and those in disease states (reviewed in 3), a

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comparative analysis of commercial proteins from diverse sources (soy, dairy, beef,

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egg, fish, and pea), or fermented or enzymatically hydrolyzed proteins has not been

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performed. Therefore, our objective was to develop a screening assay to assess the

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relative ability of various protein sources to activate MPS signaling pathways. Numerous preclinical and clinical studies have investigated the anabolic response to

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a variety of isolated protein sources including egg 4, pea 5, dairy whey and soy proteins

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6

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our understanding of the muscle health benefits arising from the consumption of dietary

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proteins. However, screening a wide variety of protein sources for their benefits in

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animal or human studies is not practicable as the number of intervention arms, and thus

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protein sources, are limited. To address these shortcomings, we developed a cell-

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based screening assay that can systematically interrogate a large and diverse set of

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dietary proteins for their ability to stimulate muscle protein signaling pathways. This in

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vitro cell-based assay allows for higher-throughput screening of a large and highly

. The use of traditional animal models and human subjects is paramount to advancing



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diverse set of dietary proteins compared with in vivo models and does so under

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physiologically relevant conditions.

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We employed murine C2C12 myoblasts differentiated into multinucleated myotubes

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for our cell-based screening assay, which permitted us to investigate the activation of

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the mechanistic/mammalian target of rapamycin (mTORC1) signaling pathway 7. There

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are two known mTOR complexes, mTORC1 and mTORC2 8, however, mTORC1 is the

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central molecular pathway involved with triggering MPS and is activated by a variety of

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stimuli including but not limited to resistance exercise 9, insulin 10, and dietary amino

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acids, specifically leucine 11. Receiving signals from many intracellular and extracellular

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signals, mTORC1 serves as the central mediator for the phosphorylation and

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subsequent activation of several downstream targets that initiate MPS: eukaryotic

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translation initiation factor 4E binding protein 1 (eIF4EBP1), S6 ribosomal protein

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(S6RP), and ribosomal protein S6 kinase, polypeptide 1 (p70S7K) 3. While the role of

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mTORC2 is not as well understood, in vivo studies suggest that mTORC2 plays a

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critical role in glycolysis and lipogenesis 12.

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Dietary proteins are hydrolyzed by acid and enzymes as they undergo digestion in

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the gastrointestinal tract after consumption, after which small peptides and amino acids

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are absorbed through the lumen of the intestines and systemically delivered to cells

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throughout the body 13. In order to model in vivo digestion, enzymes found in the

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digestive system were used in an in vitro simulated gastric digestion (SGD) system to

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hydrolyze proteins to mimic the complex mixture of peptides and amino acids delivered

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to cells in the gastrointestinal epithelium and ultimately to the muscle cells themselves

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14-15

. Proteins screened in our assay were all subject to SGD prior to being tested for 4 ACS Paragon Plus Environment

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their ability to stimulate mTORC1 mediated protein synthesis. The relative activation of

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the mTORC1 pathway in response to incubation with various SGD proteins was

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assessed by measuring the activation (phosphorylation) of p70S6K(Thr389). This

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screening tool was then used to systematically evaluate diverse commercial proteins,

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experimental soy protein hydrolysates (ESHs), and other non-commercial protein

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sources that have been differentially processed, for their ability to activate mTORC1

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signaling.

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A secondary objective of these studies was to determine whether treatment of

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proteins with food enzymes prior to simulated gut digestion could influence the ability of

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the proteins to stimulate MPS. For this purpose, we focused on soy protein. Intact (non-

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hydrolyzed) soy protein is produced by the extraction and subsequent purification of the

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total protein from soybeans (~40% of dry weight) 16. The use of food-grade proteolytic

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enzymes during the processing of intact soy protein yields protein hydrolysates, which

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have varying amounts of peptides of variable lengths 17. Potentially bioactive peptide

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fractions may be generated in protein hydrolysates and these have been proposed to be

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associated with numerous human health benefits 18 and may provide an enriched

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source of small peptides that can be absorbed more readily for MPS 19.

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The anticipated outcome of screening numerous commercial proteins and non-

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commercial soy protein hydrolysates was to provide more insights as to what

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characteristics of the proteins were associated with increased stimulation of the

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mTORC1 pathway based on the protein composition itself, i.e. in the absence of

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variables such as digestion rate, etc. We also wished to demonstrate the utility of the



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cell-based screening assay for assessing the mTORC1 stimulatory effect of novel

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protein sources and or other dietary components in the future.

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Materials and Methods

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Chemicals. Dulbecco’s Modified Eagle Medium (DMEM) growth media, Minimal

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Essential Medium α (MEMα), Dulbecco’s Phosphate-Buffered Saline (DPBS), heat-

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inactivated fetal bovine serum, horse serum, Hank’s Balanced Salt Solution (HBSS), 1X

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antibiotic-antimycotic (Gibco® 15240-062), rat tail Collagen I - coated 24-well plates and

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Pierce™ BCA Protein Assay Kits were obtained from Thermo Fisher (Waltham, MA).

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Porcine insulin, porcine pepsin, porcine pancreatin (8X USP) (Sigma P7545), O-

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pthaldialdehyde (OPA) and sodium dodecyl sulfate were obtained from Sigma-Aldrich

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(St. Louis, MO). Meso Scale Discovery phosphoprotein assays (Whole Cell Lysate Kits)

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were obtained from Meso Scale Diagnostics, LLC (Rockville, MD).

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C2C12 cell culture and screening assay. Approximately 1.8x105 C2C12 cells (ATCC,

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CRL-1772) were thawed and expanded in 150 cm2 filtered tissue culture flasks (TPP®,

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Techno Plastic Products AG, Sigma-Aldrich, St. Louis) containing Dulbecco’s Modified

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Eagle Medium (DMEM) growth media (4.5 g/L D-glucose, 584 mg/L L-Glutamine, 110

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mg/L Sodium Pyruvate) supplemented with 10% (vol/vol) heat inactivated fetal bovine

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serum and 1X antibiotic-antimycotic at 37°C, 5% CO2. Undifferentiated C2C12

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myoblasts were then seeded out at a density of 7.0x104 cells/well onto rat tail Collagen I

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- coated 24-well plates. The following day, DMEM growth media was replaced with

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DMEM differentiation media I (DMEM media (as above) supplemented with 5% (vol/vol)

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horse serum and 1X antibiotic-antimycotic) for 24 hrs and then replaced with DMEM


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differentiation media II (DMEM media supplemented with 2% (vol/vol) horse serum and

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1X antibiotic-antimycotic) for 66 hrs. DMEM differentiation media II was refreshed 24

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hrs prior to the start of the cell-based screening. In the morning, just prior to

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experimental treatments, cells were serum starved for 4 hrs in Minimal Essential

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Medium α (MEMα) followed by a 1 hr amino acid starvation in Hank’s Balanced Salt

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Solution (HBSS). After serum and amino acid starvation, C2C12 cells were treated with

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SGD proteins in HBSS for 30 min at 1 mg/mL final concentration containing 2 nM

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porcine insulin. Cells incubated with 2 nM porcine insulin in HBSS alone served as

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baseline for the assay. A maximum (20 µM) porcine insulin control (Max Insulin) was

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included in each assay as a positive control reflecting maximum MPS. Cells were

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briefly rinsed in 300 µL ice cold Dulbecco’s Phosphate-Buffered Saline (DPBS) and

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immediately placed on ice. Cells were lysed with 100 µL ice cold complete lysis buffer

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prepared according to directions from Meso Scale Diagnostics. Cellular lysates were

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stored at -80°C.

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Simulated gastric digestion (SGD). SGD was performed using similar methods

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previously described 14 with the following modifications. Briefly, 2.5 g (total dry weight)

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of each of the dietary proteins was suspended in 50 mL of Milli-Q H2O in sterilized 125

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mL Erlenmeyer flasks. The pH was adjusted to 2.3 with pre-filtered 6N HCL and 625

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U/mL of porcine pepsin and 1% V/V antibiotic/antimycotic solution were added and

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samples were incubated for 1 hr at 37°C with gentle agitation. Following pepsin

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digestion, NaOH was added to adjust the pH to 8.0. Following pH adjustment, 0.5%

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W/W porcine pancreatin was added to each sample. Samples were incubated for 4 hr

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at 37°C with gentle agitation. Samples were heat-inactivated at 95°C for 5 min, frozen 7 ACS Paragon Plus Environment


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at -80°C and lyophilized the following day. One mL of pre-and post-pepsin and

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pancreatin digested material were saved to determine degree of hydrolysis. Lyophilized

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proteins were rehydrated in HBSS and centrifuged for 30 min at 4°C at 16,000 x g to

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isolate the soluble protein fraction. After centrifugation, the soluble fraction were sterile

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filtered (0.2 µm) and stored at -80°C.

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Degree of hydrolysis determination. O-pthaldialdehyde (OPA) was used to determine

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the degree of hydrolysis for our SGD proteins using previously described methods 15, 20.

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Briefly, proteins were subjected to total acid hydrolysis (control) by incubating 10 mg

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protein in 6N HCL at 110°C overnight. The following day, total acid hydrolyzed proteins

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were neutralized with 6N NaOH (VWR International, Radnor, PA) and were sterile

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filtered. Pre-and post-digested (SGD), as well as total acid hydrolyzed proteins were

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centrifuged at 10,000 x g for 10 min to pellet any insoluble material. The resulting

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soluble protein fractions were combined with the OPA working solution [OPA hydrated

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in ethanol at 4% (w/v) and added to a 1% (w/v) solution of sodium dodecyl sulfate

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(SDS), 10 mM sodium tetraborate and 5 mM dithiothreitol in Milli-Q H2O to achieve a

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final OPA concentration of 0.08%]. Directly after mixing protein fractions, absorbance

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was measured at 340 nm in a microplate reader (BioTek, Winooski, VT) using a UV-

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transparent cuvette. The DH was calculated for each time point as a percent of cleaved

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peptide bonds relative to the total acid hydrolyzed protein fraction for each SGD protein.

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NPAL methods. Amino acid analyses were performed at Nestle Purina Analytical Labs

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(NPAL), St. Louis, MO.


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Protein concentration measurements and biomarker analyses. Pierce™ BCA Protein

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Assay Kits were used to determine protein concentrations according to manufacturer’s

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directions. Meso Scale Diagnostics phosphoprotein assays were used to measure the

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amount of phosphorylated proteins present in the sample according to manufacturer’s

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directions. The following assays were run: (Phospho-p70S6K (Thr 389), Phospho

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S6RP (Ser240/244)/Total S6RP, phospho-4E-BP1 (Thr37/46), and Phospho mTOR

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(Ser2448)/Total mTOR. Cell lysates from each protein treatment were screened in

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duplicate in any given assay. Data from single or multiple assays for each protein were

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expressed as box plots providing visualization of the median (middle line), upper and

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lower quartiles for each protein tested.

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Experimental Soy protein hydrolysate generation. Soy protein was isolated from

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defatted soy flakes by a conventional isoelectric precipitation method. The isolated

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protein was diluted to 10% solids and treated with different Generally Recognized As

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Safe (GRAS) approved enzymes at various concentrations for 60 min followed by

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inactivation with heat at 82°C for 5 min, the mixture was further subjected to ultra-high-

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temperature (UHT) sterilization and spray dried. The enzymes used are products of

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DuPont™ Danisco® (except the Bromelain) and are denoted by letters as follows: A,

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mixture of Protex®6L, Protex®7L and an exopeptidase; B, glutamyl endopeptidase 1; C,

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glutamyl endopeptidase 2; D, Multifect®PR14L; E, FoodPro®51; F, Grindamyl®PR43;

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G, Grindamyl®59; H, Bromelain; I, Protex®7L; J, Protex®15L; K, Protex®50FP; L,

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Protex®26L; M, Protex®6L; N, Protex®6L (using membrane separated soy protein

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instead of standard isolate as substrate).



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Statistical methods. Data from duplicates were averaged for each protein. Unequal

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sample sizes for each protein (replicate assays), were accounted for by unweighting

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individual proteins within each protein grouping. Analysis of variance (ANOVA) was

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conducted on data expressed as % Max Insulin (bioactivity) to test for mean differences

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among proteins. Commercial and experimental proteins were analyzed separately. In

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addition, ANOVA was used to detect differences among commercial protein groups

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(beef, dairy, egg, fish, pea, soy, and soy whey). Finally, more specific analyses were

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conducted with ANOVAs to examine proteins within each group. Following ANOVAs,

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when a significant effect was found, Tukey HSD pairwise comparisons were conducted

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to compare the proteins (or groups) to one another. To compare each protein to a

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control (Max Insulin), Dunnett’s test was also conducted. Data are represented as

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means ± SEMs unless noted.

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Results

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Degree of Protein Hydrolysis after Simulated Gut Digestion

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A list of commercial proteins used in the cell-based screening assay is provided in

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Supplemental Table 1. In order to mimic in vivo conditions, commercial and

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experimental soy hydrolysate proteins were treated with digestive enzymes using an

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established model to recapitulate conditions present in the stomach (pepsin) and upper

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gastrointestinal tract (pancreatin) 14-15. All SGD proteins were analyzed before, during

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and after each stage of SGD to determine their degree of proteolytic degradation, or

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percent degree of hydrolysis (%DH) relative to the non-hydrolyzed protein 20. There


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was an increase in %DH following both pepsin and pancreatin treatments relative to

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predigested samples for all proteins subjected to SGD (Supplemental Table 2).

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Determination of Conditions and Outcome Biomarker for C2C12 Myotube Model

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In order to investigate the response of C2C12 cells in response to SGD protein

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treatments, assay conditions were similar to those previously described 7, 21. C2C12

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myotubes were co-treated with SGD proteins plus insulin (2 nM) to mimic insulin release

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that occurs in vivo 22. The SGD protein treatment concentration of 1 mg/mL was

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empirically selected based on preliminary dose response experiments which indicated

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that this concentration maximally stimulated mTORC1 activation in response to protein

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and insulin treatments relative to baseline. Initial findings revealed that phosphorylated

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p70S6K (phos-p70S6K(Thr389)) exhibited the greatest dynamic range of stimulation in

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response to our control soy protein treatment and was subsequently used as the

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primary determinant of mTORC1 activity (phospho S6RP, phospho-4E-BP1 and

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phospho mTOR were also measured, data not shown). Overall mTORC1 activation in

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response to SGD protein treatments were reported as phos-p70S6K(Thr389) activation

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normalized to the total protein per treatment well. All values were then expressed as a

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percent of the Max Insulin positive control comparator within each assay.

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Characterization of Mechanistic Signaling and Response Consistency in C2C12

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Myotube Model

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Additional experiments were conducted to validate the functionality of the assay and

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to confirm that the protein test articles and insulin treatments directly stimulated

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mTORC1 signaling in the cell-based assay. Addition of 2 nM insulin to the C2C12 cells 11 ACS Paragon Plus Environment


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with the SGD proteins resulted in an increase in mTORC1 activation compared to

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treatment with insulin (2 nM) or SGD protein treatments alone (Figure 1). Consistent

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with previous findings, these results suggest that insulin and SGD protein treatments

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independently activate mTORC1 signaling 23. In order to identify the maximum

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mTORC1 activation response, C2C12 cells were co-treated with a maximum dose of 20

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µM insulin and SGD proteins. Determination of the maximum mTORC1 response

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provided key insight into the absolute range of achievable mTORC1 stimulation and

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assurance that the stimulatory activity of the SGD proteins was not limited in the assay.

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The addition of Rapamycin, a known inhibitor of mTOR signaling, resulted in a loss of

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mTORC1 activation (Figure1) which suggests that the insulin and SGD proteins are

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directly signaling through the mTORC1 pathway 24. In order to track assay-to-assay

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variability, a SGD protein (soy protein 9) was used as a “quality control” protein on each

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plate and its stimulatory activity was monitored for consistency throughout the entirety of

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study.

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Although all essential amino acids have been shown to stimulate mTORC1 to some

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degree, leucine demonstrates much greater potency 7. We compared co-treatment with

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leucine (5 mM) and insulin to the SGD soy protein 9 plus insulin treatments to determine

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the specific contribution of leucine alone to mTORC1 stimulation (Supplemental Figure

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1). The overall increase in mTORC1 activation from SGD protein soy protein 9 (which

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provided no more than 0.6 mM leucine) plus 2 nM insulin, compared to 5 mM leucine

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and 2 nM insulin alone suggest that factors other than leucine present in the SGD

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protein treated wells contribute to activation of mTORC1 and presumably MPS.

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C2C12 Myotube Responses to Diverse Commercial Proteins. 12 ACS Paragon Plus Environment

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The median mTORC1 stimulatory response for all proteins tested are depicted as

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box plots with median responses indicated by the midlines, the upper and lower edges

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of the boxes representing the upper and lower quartiles, and the total range for all data

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points represented by the error bars (whiskers) (Figure 2). Control treatment with HBSS

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alone served as the untreated baseline. The addition of insulin (2 nM) provided a

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modest increase in mTORC1 signaling (16.0 ± 0.8% (mean ± SEM) of Max Insulin)

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when compared to the HBSS baseline response (9.5±0.5% of Max Insulin). All SGD

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commercial protein plus insulin (2 nM) treatments were grouped based on their source

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and each representative’s mTORC1 signaling response is reported as a percentage of

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the Max Insulin response. The average response for all commercial proteins screened

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(58.1±15.0% of max insulin) was used as the pooled mean mTORC1 response (Figure

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1 and Figure 2, solid line). The pooled mean value served as a benchmark and a

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significantly different stimulatory response was considered as mTORC1 activation at

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two standard deviations above the pooled mean mTORC1 response (100.4% of max

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insulin) (Figure 1 and Figure 2, dashed line). A dynamic range in mTORC1 signaling

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stimulation was observed in response to SGD proteins depending on the protein source

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and commercial processing conditions (Figure 2A). Commercially available fish protein

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(Fish Protein 2, 112.5% of max insulin), dairy whey conjugated with leucine (Dairy Whey

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Protein 3, 86.6 ± 2.3%), beef protein isolate (Beef Protein 2, 83.8 ± 5.4%), and soy

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protein concentrate (soy protein 8, 77.4 ± 2.9%) showed the highest degree of

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mTORC1 signaling compared to all other commercial proteins screened. Interestingly,

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proteins from the same source activated mTORC1 differentially. These results suggest



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that the source and the commercial processing of the dietary protein source are critical

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determinants of bioactivity with regards to mTORC1 stimulation.

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C2C12 Myotube Responses to Non-Commercial Soy Protein Hydrolysates

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A body of research supports the idea that hydrolyzed proteins may activate MPS

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more effectively than non-hydrolyzed proteins 19. We hypothesized that differential

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processing and subsequent enzymatic hydrolysis of proteins would produce protein

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hydrolysates with unique peptides profiles that would stimulate mTORC1 with varying

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efficacy. A series of food-grade enzymes (A-M) were selected and used at various

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enzyme:protein concentrations in a commercially scalable process to generate a

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diverse library of novel hydrolysates from soy protein. Experimental soy protein

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hydrolysates (ESH) were then subjected to SGD and subsequently screened for

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mTORC1 stimulatory activity in the cell-based assay (Figure 2B). Differences in the

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degree of mTORC1 stimulation by different ESH’s suggest that the hydrolysis conditions

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(dose and specific enzyme used to create the hydrolysates) are determinants of their

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activity. ESH-M protein elicited the highest mTORC1 activation response compared to

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all other ESHs screened. Furthermore, we investigated whether there was any potential

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synergistic activation of mTORC1 by hydrolyzing soy protein 8 (highest mTORC1

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activation of all commercial soy proteins screened) with ESH-M enzyme at varying

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enzyme:protein ratios (Figure 2B, ESH-N1-5). Results from the screening assay show

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that bioactivity for the ESH-N treatments were equivalent to the commercial soy protein

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8 treatment (Figure 2B).


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Two non-commercial (experimental) soy protein hydrolysates that were extensively

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hydrolyzed (ESH-A1-4 (whole hydrolysate) and ASP1-6 (latter protein hydrolysates

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consisted of the soluble fraction after centrifugation)) were found to activate mTORC1

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signaling below the average response for all commercial proteins screened and suggest

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that extensively hydrolyzed soy proteins are less effective at activating mTORC1

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compared to less hydrolyzed or intact protein preparations after all were subjected to

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simulated gut digestion (Figure 2B). The ASP sample amino acid profiles were deficient

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in some amino acids compared to the whole soy protein hydrolysate (data not shown)

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which may also account for the lower mTORC1 stimulation.

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Three different preparations of a recombinant protein (RP1-3), were produced

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through fermentation of non-pathogenic Trichoderma reesei and purified, were also

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subjected to SGD and tested for mTORC1 stimulatory activity. The observed bioactivity

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for all three preparations of this RP (RP1-3) resulted in the activation of mTORC1 that

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was equivalent to the average response observed for all other commercial proteins

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screened (Figure 2B). This protein, representing a single gene product expressed at

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high levels in Trichoderma reesei, presents an interesting opportunity to engineer

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dietary proteins with custom peptide sequences and ideally, enhanced bioactivity.

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An ANOVA analysis on mTORC1 stimulatory activity showed a significant protein

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treatment effect among individual commercial proteins (P=0.0001), commercial proteins

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grouped by source (P=0.0006), commercial only soy proteins (P=0.0001), individual

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experimental proteins (P=0.0001), and ESH-M treatments (P=0.0001). Subsequently,

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Tukey HSD pairwise comparisons following each significant ANOVA determined which

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proteins were significantly different from one another. Interestingly, numerous high15 ACS Paragon Plus Environment


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quality proteins from diverse sources (fish, dairy, beef and soy) maximally activated

348

mTORC1 to a similar degree compared to all other commercial proteins screened with

349

no statistically significant differences observed between the proteins demonstrating

350

maximal mTORC1 stimulation regardless of source (Supplemental Table 3 and 4).

351

Tukey HSD pairwise comparisons of commercial soy proteins revealed that the soy

352

protein concentrate (soy protein 8) had an average mTORC1 activating response that

353

was higher, but that did not achieve statistical significance, compared with all other

354

commercially available soy proteins screened in the cell-based assay (Supplemental

355

Table 5). Similarly, statistical analyses of all the experimental proteins identified certain

356

soy protein hydrolysates with significantly greater mTORC1 stimulatory activity

357

compared with others, with one hydrolysate generated with enzyme M as have the

358

highest mTORC1 stimulatory activity compared with the other experimental proteins and

359

soy hydrolysates (Supplemental Table 6).

360

Amino Acid Composition of Subset of Commercial and Experimental Proteins

361

The branched chain amino acids (BCAAs), valine, isoleucine and specifically

362

leucine, stimulate mTORC1 signaling and MPS 25. To investigate associations between

363

protein amino acid composition and the ability to activate mTORC1, amino acid

364

analyses were conducted for ten proteins screened in the cell-based assay. These

365

proteins were selected as representatives of proteins originating from diverse sources,

366

production methods and eliciting variable mTORC1 activation in the C2C12 cell-based

367

assay. Amino acid compositions for each of the ten proteins are shown in Table 1 with

368

the BCAAs highlighted. Regression analyses indicate that neither leucine nor the total

369

BCAA predict mTORC1 activating bioactivity (Figure 3 A-B). These results suggest that 16 ACS Paragon Plus Environment

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the high quality proteins analyzed in our assay stimulate mTORC1 to a similar degree

371

and that factors in addition to leucine or BCAA concentrations are necessary to

372

maximally stimulate mTORC1 signaling.

373

Discussion

374

Dietary proteins are critical macronutrients that can function as key stimulators of

375

MPS via amino acid-sensitive activation of the mTORC1 pathway and its downstream

376

target proteins. Upon digestion in the gut, dietary proteins are sources of peptides with

377

potential bioactivity as well as free amino acids and bioactive compounds. Results from

378

numerous human clinical trials indicate that there are significant muscle health benefits

379

from both exercise and dietary protein intake, both of which activate mTORC1 signaling

380

and MPS and can attenuate muscle wasting 26. Understanding whether and how

381

different proteins stimulate mTORC1 and MPS is important for making relevant dietary

382

protein choices for individuals at all stages of life, but perhaps even more important for

383

those experiencing or at risk of muscle wasting, such as elderly or hospitalized subjects.

384

In the present study, a series of dietary proteins from various sources and processing

385

conditions were screened for their ability to activate mTORC1 signaling. Our results

386

indicate that most high-quality proteins activate mTORC1 signaling to a similar degree

387

with no significant difference between soy, dairy, beef or egg protein. Furthermore,

388

there was no correlation between the amount of leucine or BCAAs and the degree of

389

mTORC1 activation in a subset of soy, dairy, beef and fish protein analyzed.

390 391

Efficacy of our dietary protein treatments to stimulate mTORC1 signaling was assessed by measuring the phosphorylation of the downstream signaling molecule p70-



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S6K1. Many studies have identified this critical signaling molecule as a key mediator in

393

translating activation of mTORC1 to activation of muscle protein synthesis (MPS) 8, 27-

394

29

395

assay would be expected to translate into activation of MPS. It is also noteworthy that

396

hyperactivation of mTORC1 signaling may not be desirable for individuals already in

397

specific disease states and may contribute to the pathology of neurological diseases,

398

type II diabetes and cancer 30-31, therefore understanding factors that contribute to

399

hyperactivation is also of interest. Results from our screening efforts show that diverse

400

dietary proteins elicit a broad range of mTORC1 activation in vitro, ranging from

401

approximately 20% to over 100% activation of mTORC1 signaling relative to the

402

maximal stimulation achievable in the assay (Max Insulin control) (Figure 1A,B).

403

Interestingly, differential processing of proteins from the same source resulted in similar

404

diversity of mTORC1 stimulatory activity (e.g dairy and soy proteins in Figure 2A and

405

the differentially hydrolyzed soy proteins in Figure 2B). Further studies are needed to

406

understand how the initial processing of the food protein may affect the products of

407

simulated gut digestion and their ability to stimulate mTORC1.

408

. Thus, the observed stimulation of mTORC1 by the various proteins in the C2C12

Enzymatic hydrolysis of proteins generates di and-tripeptides, which are absorbed at

409

a greater rate than individual amino acids 19, and other short peptide sequences that

410

may have unique bioactivities in vivo. Such bioactive peptides have been identified from

411

various protein sources including, fish 32, human milk 33, and soy 34. This study also

412

investigated whether using various food endo-and exopeptidases could impact the

413

mTORC1 stimulatory bioactivity of soy protein. Our results indicate that both the

414

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factors for the protein’s overall ability to induce p70S6K phosphorylation in vitro (Figure

416

2B). Previous research demonstrated that soy proteins hydrolyzed with food enzymes

417

prior to SGD were more rapidly absorbed compared to non-hydrolyzed soy proteins

418

subjected to SGD 15. The current study did not evaluate the rate of uptake of peptides

419

and/or amino acids from the hydrolysates, therefore we cannot know whether different

420

rates of absorption and/or the bioactive nature of the peptides generated by the different

421

enzymes and conditions itself, may have played a role in the differences in mTORC1

422

stimulation seen with the different soy protein hydrolysates. It is worthwhile noting that

423

the acid soluble peptides (ASP samples) separated from the whole soy hydrolysates did

424

not stimulate mTORC1 to the same degree as the unfractionated soy hydrolysates

425

(Figure 2B).

426

The amino acid leucine has been extensively investigated for its ability to activate

427

MPS. Leucine activates MPS through the rapamycin-sensitive mTORC1 pathway 27 as

428

well as through an mTORC1-independent manner 28. Recently, intracellular proteins

429

that act as sensors for the amino acids arginine and leucine have been identified that

430

are upstream regulators of mTORC1 35-36. In addition, numerous factors besides

431

leucine have been shown to activate MPS, including glucose and the amino acids,

432

glutamine and tryptophan 37-39. We performed amino acid analyses on ten diverse and

433

differentially processed dietary proteins and to determine whether the Leu or BCAA

434

content alone could account for the proteins’ mTORC1 stimulatory activity. The

435

regression analysis shown in Figure 3 indicate that neither leucine nor the total BCAA

436

content were predictors of mTORC1 signaling activity. These data support the notion

437

that factors, in addition to leucine and total BCAA, are necessary for maximally 19 ACS Paragon Plus Environment


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activating mTORC1 3, 37. Such factors may include other amino acids, specific peptides

439

formed post-digestion and/or other bioactive components present in the dietary protein

440

ingredients.

441

Currently, non-protein components present in dietary protein sources that may

442

contribute to healthy muscle metabolism are not well understood. Legumes, specifically

443

soy, may exert health benefits through the activity of diverse bioactives that are often

444

retained in isolated protein ingredients, including phytosterols, phospholipids, fiber,

445

saponins, and isoflavones 16. Interestingly, the isoflavones found in soy have been

446

shown to inhibit MPB via the ubiquitin proteasome pathway in C2C12 cells 40. While

447

MPB signaling pathways in response to SGD protein treatments were not investigated

448

in the current study, future studies evaluating the impact of soy protein-associated

449

isoflavones on MPB may be of interest. Given our observations of differential mTORC1

450

signaling in response to a variety of protein sources, it remains to be determined

451

whether those proteins that resulted in higher mTORC1 stimulatory activity, if added in

452

combination, would lead to additive or synergistic mTORC1 stimulation. Since the

453

molecular mechanism(s) responsible for each protein’s stimulatory activity was not

454

investigated in this study, it is possible that unique components from each protein may

455

work in tandem to activate the mTORC1 pathway. Previous studies have demonstrated

456

enhanced MPS following ingestion of soy/dairy protein blends in both rats 6 and in

457

humans 41. Additional studies are required to understand the anabolic response to

458

combinations of dietary proteins.

459 460

While our model system provides a relatively quick way to assess the potential impact of dietary protein sources on muscle protein synthesis signaling, there are some 20 ACS Paragon Plus Environment

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limitations. Data are generated in an in vitro system and confirmation of any conclusions

462

through in vivo testing is required. Furthermore, we focused on the phosphorylation of

463

p70S6K, which while a critical mediator of mTORC1 signaling, is not the only signaling

464

molecule that may be differentially modulated by different protein sources. We did not

465

measure protein synthesis per se or changes in myotube diameter in this experiment,

466

therefore it is not known whether the increase in mTORC1 signaling would translate into

467

increased protein synthesis. Finally, our studies assume that much of the soluble

468

protein digests reaches the muscle cells, when in fact, protein digestion and absorption

469

of the peptides and amino acids in the gut is complex and plasma profiles of absorbed

470

nutrients may change over time.

471

To conclude, the data presented here indicates that both the protein source and

472

processing conditions can have a significant impact on the protein’s ability to activate

473

mTORC1 signaling, however, there is no significant difference in mTORC1 stimulatory

474

activity between high quality protein sources such as dairy, soy, beef and egg. Factors

475

in addition to the leucine or BCAA content of the proteins play a role in stimulating the

476

mTORC1 pathway. Additional research is needed to better understand what specific

477

factors associated with the different protein sources impact mTORC1 activation and

478

whether these factors work additively or even synergistically. Ultimately, pre-clinical and

479

human clinical trials will be essential to confirm and translate the results of such

480

investigations to practical dietary recommendations and practices.

481

Acknowledgement



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The authors are grateful to Dave Estell of DuPont Industrial Biosciences for providing

483

the samples of the recombinant protein.

484

Funding Sources

485

This study was wholly funded by DuPont Nutrition & Health.

486

Supporting Information

487

Supplemental Table 1, Sources of commercial proteins used in the cell-based screening

488

assay; Supplemental Table 2, Degree of hydrolysis for commercial and experimental

489

(non-commercial) proteins prior to simulated gut digestion and post-pepsin and post-

490

pancreatin; Supplemental Table 3, mTORC1 stimulatory activity for commercially

491

available proteins screened in the in cell-based assay; Supplemental Table 4, mTORC1

492

stimulatory activity for commercially available protein sources screen in the cell-based

493

assay; Supplemental Table 5, mTORC1 stimulatory activity for commercially available

494

soy proteins screened in the cell-based assay; Supplemental Table 6, mTORC1

495

stimulatory activity by level of activation for representative experimental soy protein

496

hydrolysates, acid soluble soy protein hydrolysates and recombinant proteins (non-

497

commercial); Supplemental Figure.1, Effect of leucine (5 mM) plus insulin (2 nM)

498

compared with SGD protein (1 mg/mL) and insulin (2 nM) treatments on the activation

499

of mTORC1 signaling in C2C12 cells.

500

501

502 22 ACS Paragon Plus Environment

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References

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16. Medic, J.; Atkinson, C.; Hurburgh, C. R., Current Knowledge in Soybean Composition. J. Am. Oil Chem. Soc. 2014, 91 (3), 363-384. 17. Ortiz, S. E. M.; Wagner, J. R., Hydrolysates of native and modified soy protein isolates: structural characteristics,, solubility and foaming properties. Food. Res. Int. 2002, 35 (6), 511518. 18. Udenigwe, C. C.; Aluko, R. E., Food protein-derived bioactive peptides: production, processing, and potential health benefits. J. Food Sci. 2012, 77 (1), R11-24. 19. Manninen, A. H., Protein hydrolysates in sports nutrition. Nutr. Metab. (Lond) 2009, 6, 38. 20. Church, F. C.; Porter, D. H.; Catignani, G. L.; Swaisgood, H. E., An o-phthalaldehyde spectrophotometric assay for proteinases. Anal. Biochem. 1985, 146 (2), 343-8. 21. Mao, X.; Zeng, X.; Wang, J.; Qiao, S., Leucine promotes leptin receptor expression in mouse C2C12 myotubes through the mTOR pathway. Mol. Biol. Rep. 2011, 38 (5), 3201-6. 22. Kalogeropoulou, D.; Lafave, L.; Schweim, K.; Gannon, M. C.; Nuttall, F. Q., Leucine, when ingested with glucose, synergistically stimulates insulin secretion and lowers blood glucose. Metab. Clin. Exp. 2008, 57 (12), 1747-52. 23. Gran, P.; Cameron-Smith, D., The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes. BMC Physiol. 2011, 11, 10. 24. Ye, L.; Varamini, B.; Lamming, D. W.; Sabatini, D. M.; Baur, J. A., Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2. Front. Gen. 2012, 3, 177. 25. Drummond, M. J.; Dreyer, H. C.; Fry, C. S.; Glynn, E. L.; Rasmussen, B. B., Nutritional and contractile regulation of human skeletal muscle protein synthesis and mTORC1 signaling. J. Appl. Physio.l (1985) 2009, 106 (4), 1374-84. 26. Denison, H. J.; Cooper, C.; Sayer, A. A.; Robinson, S. M., Prevention and optimal management of sarcopenia: a review of combined exercise and nutrition interventions to improve muscle outcomes in older people. Clin. Inter. Aging 2015, 10, 859-69. 27. Anthony, J. C.; Yoshizawa, F.; Anthony, T. G.; Vary, T. C.; Jefferson, L. S.; Kimball, S. R., Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway. J. Nutr. 2000, 130 (10), 2413-9. 28. Bolster, D. R.; Vary, T. C.; Kimball, S. R.; Jefferson, L. S., Leucine regulates translation initiation in rat skeletal muscle via enhanced eIF4G phosphorylation. J. Nutr. 2004, 134 (7), 1704-10. 29. Hornberger, T. A.; Sukhija, K. B.; Wang, X. R.; Chien, S., mTOR is the rapamycinsensitive kinase that confers mechanically-induced phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k). FEBS Lett. 2007, 581 (24), 4562-6. 30. Carunchio, I.; Curcio, L.; Pieri, M.; Pica, F.; Caioli, S.; Viscomi, M. T.; Molinari, M.; Canu, N.; Bernardi, G.; Zona, C., Increased levels of p70S6 phosphorylation in the G93A mouse model of Amyotrophic Lateral Sclerosis and in valine-exposed cortical neurons in culture. Exp. Neurol. 2010, 226 (1), 218-30. 31. Dann, S. G.; Selvaraj, A.; Thomas, G., mTOR Complex1-S6K1 signaling: at the crossroads of obesity, diabetes and cancer. Trends Mol. Med. 2007, 13 (6), 252-9. 32. Ryan, J. T.; Ross, R. P.; Bolton, D.; Fitzgerald, G. F.; Stanton, C., Bioactive peptides from muscle sources: meat and fish. Nutrients 2011, 3 (9), 765-91. 33. Wada, Y.; Lonnerdal, B., Bioactive peptides derived from human milk proteins-mechanisms of action. J. Nutr. Biochem. 2014, 25 (5), 503-14. 24 ACS Paragon Plus Environment

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34. Singh, B. P.; Vij, S.; Hati, S., Functional significance of bioactive peptides derived from soybean. Peptides 2014, 54, 171-9. 35. Wang, S.; Tsun, Z. Y.; Wolfson, R. L.; Shen, K.; Wyant, G. A.; Plovanich, M. E.; Yuan, E. D.; Jones, T. D.; Chantranupong, L.; Comb, W.; Wang, T.; Bar-Peled, L.; Zoncu, R.; Straub, C.; Kim, C.; Park, J.; Sabatini, B. L.; Sabatini, D. M., Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1. Science 2015, 347 (6218), 18894. 36. Wolfson, R. L.; Chantranupong, L.; Saxton, R. A.; Shen, K.; Scaria, S. M.; Cantor, J. R.; Sabatini, D. M., Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 2016, 351 (6268), 43-48.. 37. Dukes, A.; Davis, C.; El Refaey, M.; Upadhyay, S.; Mork, S.; Arounleut, P.; Johnson, M. H.; Hill, W. D.; Isales, C. M.; Hamrick, M. W., The aromatic amino acid tryptophan stimulates skeletal muscle IGF1/p70s6k/mTor signaling in vivo and the expression of myogenic genes in vitro. Nutrition 2015, 31 (7-8), 1018-24. 38. Jeyapalan, A. S.; Orellana, R. A.; Suryawan, A.; O'Connor, P. M.; Nguyen, H. V.; Escobar, J.; Frank, J. W.; Davis, T. A., Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mTOR-independent process. Am. J. Physiol. Endocrinol. Metab. 2007, 293 (2), E595-603. 39. Duran, R. V.; Oppliger, W.; Robitaille, A. M.; Heiserich, L.; Skendaj, R.; Gottlieb, E.; Hall, M. N., Glutaminolysis activates Rag-mTORC1 signaling. Mol. Cell 2012, 47 (3), 349-58. 40. Hirasaka, K.; Maeda, T.; Ikeda, C.; Haruna, M.; Kohno, S.; Abe, T.; Ochi, A.; Mukai, R.; Oarada, M.; Eshima-Kondo, S.; Ohno, A.; Okumura, Y.; Terao, J.; Nikawa, T., Isoflavones derived from soy beans prevent MuRF1-mediated muscle atrophy in C2C12 myotubes through SIRT1 activation. J. Nutr. Sci. Vitaminol. 2013, 59 (4), 317-24. 41. Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Fry, C. S.; Borack, M. S.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.; Rasmussen, B. B., Protein blend ingestion following resistance exercise promotes human muscle protein synthesis. J. Nutr. 2013, 143 (4), 410-6.

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TABLE 1 Amino Acid Composition for Commercial and Experimental Proteins Screened in the Cell-Based Assay AA1

BP2

DWP2

DWP3

DWP4

FP2

Ala

8.5

5.5

3.4

5.5

6.6

4.3

Arg

6.9

2.7

1.9

2.2

5.0

Asp

6.0

12.4

8.1

12.1

Glu

11.3

19.3

13.8

Gly

18.9

1.9

His

1.1

Ile

ESH-E08 ESH-G1

SP3

SP8

SP9

4.3

4.3

3.4

4.2

7.6

7.7

7.5

3.6

7.6

9.0

11.4

11.4

11.4

7.8

11.4

20.5

13.8

20.2

20.6

20.4

23.3

20.4

1.2

1.8

7.0

4.2

4.2

4.2

1.9

4.1

2.1

1.2

1.8

2.4

2.6

2.6

2.6

2.9

2.5

1.6

5.9

5.1

7.1

4.5

4.7

4.7

4.7

5.2

4.6

Leu

3.7

13.3

42.0

11.2

7.2

7.7

7.8

7.7

9.7

7.7

Lys

3.5

11.5

6.9

10.2

7.1

6.2

6.2

6.1

7.9

5.9

Met

1.1

2.5

1.4

2.4

2.7

1.3

1.3

1.3

2.9

1.3

Phe

2.4

3.9

2.1

3.3

3.8

5.3

5.4

5.4

5.1

5.3

Pro

10.8

5.7

4.9

7.1

5.0

5.5

5.4

5.4

9.9

5.5

Ser

3.0

4.1

3.4

5.1

4.7

5.0

5.1

5.0

5.6

5.0

Thr

2.1

5.3

5.0

7.9

4.7

3.8

3.8

3.8

4.5

3.7


Page 27 of 35


Roeseler DA et al.

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Tyr

1.0

4.1

2.1

3.2

3.2

3.9

3.9

3.9

5.5

3.7

Val

2.8

5.8

4.2

6.6

6.1

5.1

5.1

5.1

6.9

5.1

Trp

0.2

2.1

1.2

1.7

1.1

1.1

1.1

1.2

1.2

1.2

Cys

0.1

3.6

1.9

2.6

1.1

1.2

1.1

1.2

0.8

1.0

8.2

25.1

51.2

24.8

17.7

17.5

17.7

17.4

21.8

17.4

Total BCAA

2

Amino acid compositions for a subset of proteins prior to SGD was determined by Nestle Purina Analytical Laboratories (NPAL). 1Amino acid content expressed as g AA/ 100 g protein.

2

BCAA, isoleucine (Ile), leucine

(Leu), and valine (Val) are highlighted in grey. AA, Amino Acid; BP, Beef Protein; BCAA, Branched chain amino acids; DWP, Dairy Whey Protein; ESH, Experimental Soy Hydrolysate; FP, Fish Protein; SGD, simulated gut digestion; SP, Soy Protein



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Figure Captions Figure 1 Validation of mTORC1 bioactivity in mouse C2C12 myotubes showing bioactivity of C2C12 cells treated with insulin plus SGD protein treatment. Values represent means ± SEMs. SGD control soy protein 9 or dairy whey protein 2 treatments demonstrate an increase in mTORC1 activation when tested alone or in combination with insulin (2 nM). Rapamycin treatment (200 nM) plus SGD protein and insulin (2 nM) treatments suggest that both treatments and the subsequent activation of mTORC1 signaling are rapamycin sensitive. Figure 2 Percent of maximal insulin stimulation of mTORC1 signaling target, phos-p70S6K(Thr389) following SGD protein treatments in mouse C2C12 myotubes. Box plots capture the median (mid line), upper and lower quartiles (boxes) and variability (whiskers) of A) commercial protein treatments or B) Experimental soy protein treatments. Overall bioactivity is reported as the activation of phos-p70S6K relative to total protein and taken as a percentage of the max insulin (20 µM) positive control comparator. The mean bioactivity response for all commercial proteins (58.1% of max response, solid line) and two standard deviations above the mean (100.4% of max response, horizontal dashed line) are shown. HBSS = Hanks Balanced Salt Solution, MPI = Milk Protein Isolate, WPC = Whey Protein Concentrate, SWP = Soy Whey Protein, ASP = Acidsolubilized Soy Peptides (produced with alcalase enzyme hydrolysis)


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Figure 3 Regression analyses to determine if total leucine or BCAA predict mTORC1 bioactivity for a subset of proteins from diverse sources screened in the cell-based screening assay. A.) Bioactivity was compared to total leucine or B) total BCAA content. Amino acid (AA) content expressed as (g AA /100 g protein).

♦ = Beef Protein; ▲ = Fish Protein; ■ = Dairy Protein; ● = Soy Protein



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Figure 1


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Figure 2

A



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Figure 2

B


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Figure 3



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C2C12 Myotubes

Murine C2C12 myoblasts Differentiate (6 days) PO4 Thr389

Day of Assay p70S6K Serum-Starve 4 hr

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Digested Protein Incubation 30 mins

ACS Paragon Plus Environment

Lyse Cells

Muscle Protein Signaling in C2C12 Cells Is Stimulated to Similar

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