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Introduction
Myogenic satellite cells (SC) are postnatal skeletal muscle stem cells (1, 2). Without the activity of myogenic SC, postnatal skeletal muscle hyperplasia, hypertrophy, or repair would be impossible (3). Satellite cell fusion (4–6) with adjacent myofibers provides the nuclei required for regulating myofibrillar protein accretion (7), and the cellularity of these cells is required for replacing damaged myofibers (1). The history of the development of the ovine satellite cell system, in vitro, (1, 4–10), and the rationale for its use in the types of studies described, herein, have been reported (11–18).
Understanding the linkage between SC activity and dietary components is a timely area of research. Whether simply elucidating the influence of dietary constituents on satellite cell proliferation and differentiation, or generating complex knowledge regarding the influence of diet on gene regulation (nutrigenomics; 19) or metabolism (metabolomics; 19), information gained in this arena will help us understand the complex regulation of stem cells found in skeletal muscle. As a consequence, the present study was undertaken to gain additional knowledge about the mechanisms through which individual dietary supplements regulate SC activity in vitro.
Materials and Methods
Materials
The compounds were obtained from companies that supply manufacturers of dietary supplement products (Table 1). Dulbecco’s Modified Eagle Medium (DMEM), horse serum (HS), penicillin/streptomycin, and gentamicin were purchased from Invitrogen/Life Technologies. Pigskin gelatin (porcine skin, type A), Giemsa stain (Accustain), trypsin, KCl, KH2PO4, NaCl, Na2HPO4, and Na2 EDTA•2H2O were purchased from Sigma Chemical Company. The cell culture plates and flasks were purchased from Nalge NuncTM.
Cells
The satellite cells used for the present studies were exactly as described previously (13, 14). The basal medium for proliferation consisted of DMEM+2% HS whereas the differentiation medium consisted of DMEM+0.5% HS. The treatment compounds were added to the basal medium at four separate levels. The treatment medium was changed every 24 hr until a pre-determined endpoint was reached (96 h for proliferation, 120 h for differentiation). At this time, the cultures were removed from incubation, fixed with methanol, stained with Giemsa, and then counted to determine the effects of treatment on cell proliferation and/or differentiation (13, 14).
Test compounds
Water soluble compounds were added to plain DMEM to make a 0.1% stock solution. Six compounds (Evodiamine, Diachrome, CarnisheildTM B94, SoLiteTM Blend, Humanofort 17-Keto SulfateTM, and Vita Blue Wild Blueberry Extract) had high particulate matter after suspending in DMEM and were centrifuged for 5 min (SoLiteTM Blend, Humanofort 17-Keto SulfateTM, CarnisheildTM B94, and Vita Blue Wild Blueberry Extract) or 10 min (Evodiamine, Diachrome) at 2700 rpm (1500×G using a horizontal rotor; Beckman TJ-6 centrifuge) and the supernatant retained. The pH of all the stock solutions was returned to 7.08 and sterile-filtered first through a 0.45 μm pore syringe filter to remove larger particulate matter and then sterile-filtered through a 0.2 μm pore syringe filter. For all compounds, the first treatment level was a 1 : 10 dilution of the stock solution and the remainder of the treatments consisted of three 1 : 100 serial dilutions. The control for each compound consisted of basal media without the test compound.
Fatty acids and oils were either added to DMSO or to 100% ethanol to aid in solubility. Marinol D50 Omega 3, Clarinol G-50, and NKOTM 100% Krill Oil were suspended in DMSO (2.5 ml compound in 5 ml DMSO) to make a saturated solution. Clarinol G-50 and NKOTM were removed from the capsule before adding to the DMSO. The suspensions were filtered through a 0.45 μm pore syringe filter and then sterile-filtered through a 0.2 μm pore syringe filter rated for DMSO to make a stock solution. The first treatment level was a 1 : 10 dilution of the stock medium. The remainder of the treatments consisted of three serial 1 : 100 dilutions. It was determined that the highest treatment level contained 0.067 μl of DMSO/ml medium; therefore this amount of DMSO was added to the controls. The controls consisted of basal media+0.67 μl DMSO/ml basal medium. Clarinol G-80 was added to 100% ethanol to yield a 10% stock solution and sterile-filtered through a 0.2 μm pore syringe filter. The solution was returned to pH 7.08. The first treatment was a 1 : 100 dilution of the stock (for a 0.1% solution) in basal medium, and the remaining treatments were three serial 1 : 100 dilutions. The contents of one CLA Ultra Lean capsule were removed with a needle and syringe, added to 7.8 ml of 100% ethanol, and filtered through a 0.2 μm pore syringe filter to make a 5% stock solution. The first treatment is a 1 : 50 dilution of this stock into basal medium (for a 0.1% solution), and the remaining treatments were three 1 : 100 serial dilutions. BioAstin Oleoresin 5% Astaxanthin/95% safflower oil was added to 100% ethanol for a stock solution and sterile filtered through a 0.2 μm pore syringe filter. The first treatment level was a 1 : 1,000 dilution of the stock, and the remaining three treatments were serial 1 : 100 dilutions. The controls for these compounds consisted of basal medium with 1 μl of 100% ethanol per milliliter of medium added. The concentrations of all the compounds ranged from below physiologically relevant to toxic levels.
Serum-free proliferation
The compounds that were determined to have a statistically significant treatment effect in serum-containing medium were selected for further testing in a basal, serum-free defined medium: ovine defined medium (ODM) for proliferation (REF) and ITT-C (insulin, transferrin, triiodothyronine, cortisol) for differentiation (18). Creatine monohydrate (CM) has been shown to affect satellite cell differentiation (18); therefore CM was added to the defined media to test for an additive effect.
Statistical analysis
All screening experiments were performed and evaluated exactly as previously described in our prior study on ergogenic compounds (18). The experiments consisted of four wells per treatment point and were performed twice for a total of eight wells per treatment. At the termination of the experiments, all the cultures were counted, data were collated and reduced, treatment differences were detected with a one-way ANOVA and post-tests were conducted using Tukey’s pairwise comparisons. The statistical analysis was performed using Graphpad Instat version 3.05. The data was presented as Mean±SE and considered significant when (p<0.05).
Results
Proliferation and Differentiation
The morphology of all the cells was normal. The highest levels of fatty acid compounds caused cell death. The results of the proliferation assays are summarized in Table 2. Significant increases in cell numbers compared to control values (p<0.05) were found in 10 compounds: Disodium 5′-inosinate, Disodium guanosine 5′-monophosphate, Disodium inosine 5′-monophosphate, L-citrulline, ATP, Caffeine anhydrous powder, Ecdysterone, Humanofort 17-keto sulfate, CLA Ultra Lean, Clarinol G-80. For differentiation, the morphology of all the cells was normal. The highest levels of fatty acid compounds caused cell death. Significant increases in SC differentiation compared to control values (p<0.05) were found for four compounds: disodium 5′-guanylate, disodium inosine 5′-monophosphate, taurine, and disodium inosinate I003-XX-SOD-PD-RRR.
Discussion
In the present study, we attempted to gain knowledge about the regulation of postnatal skeletal muscle stem cells (3, 7) as influenced by a variety of different nutraceutical agents (18) using an assay system previously validated (11–18). Satellite cells are important for normal skeletal muscle growth (increase in muscle mass) and repair (3, 7). Interest in stimulating muscle mass encompasses three major areas: 1) muscle wasting disorders; 2) sarcopenia of aging and 3) athletic or physical performance.
In addition to consuming adequate caloric and protein intake during resistance training (20), creatine supplementation is generally regarded as effective for increasing muscle mass and strength (20, 21). Previous work with muscle SCs has found that creatine had consistent effects on satellite cell differentiation in vitro (7), which may partly explain the ability of exogenous creatine to enhance muscle mass and strength. Other compounds, mostly nucleic acid salts and taurine, have been shown to affect muscle satellite cell proliferation and/or differentiation in this study (Table 2), adding to a list of nutrients found in dietary supplements that have the potential to positively affect muscle mass (2, 5–8). Other nutrients with effects on satellite cells included certain fatty acids, such as conjugated linoleic acid (CLA). Fatty acids (FA) and FA derivatives are thought to exert effects through gene regulation (22), perhaps mediated via the nuclear protein peroxisome proliferator activated receptor (PPAR) to mediate the differentiation and subsequent filling of adipocytes (23). Similarly, for muscle cells linoleic acid has been determined to enhance the formation of myotubes from isolated myogenic satellite cells (24) and in L6 myoblast cultures (25). Additional experimentation with conjugated linoleic acids (CLA) and L6 myoblasts showed both a proliferative and/or a differentiative effect depending on the dose of CLA and the isomer of CLA to which the cells were exposed (25), whereas protein complexes like Humanofort (a mixture of growth factors and other proteins; (26) had only minor effects on SC proliferation.
What is the ramification of individual dietary components altering the activity of satellite cells in vitro? The effects in vitro may/may not be operative in vivo after oral administration. Digestion, absorption, and metabolism all influence the concentrations and identities of ingested nutrients presented to satellite cells. The relevance of sheep muscle to human muscle is also another variable to consider, but a variety of different animals have been used for satellite cell research with a final reference towards providing knowledge parallel to human physiology (3). Nevertheless, despite limitations, the research presented here is a starting point for the evaluation of dietary supplement ingredients as legitimate agents for enhancing muscle activity.
The effect on muscle SCs is one possible mechanism for encouraging muscular hypertrophy. In theory, affecting SCs should also have activity in non-athletic muscle activity, such as muscular wasting or sarcopenia of aging. In general, the results from muscle SCs on proliferation and differentiation are compatible with other data for compounds with the potential utility to support muscle repair, hypertrophy and hyperplasia. As the world population increases, so will infirmity and decreased functional ability of the elderly and infirmed. Knowledge of which compounds might aid treating muscle wasting, or which compounds may enhance future stem cell therapies will be increasingly important. The logical place to begin looking for treatments for maintaining mobility and load-bearing capability is the stem cells (satellite cells) of skeletal muscle. Collectively, these data provide basic knowledge about stem cells in skeletal muscle and suggest that satellite cells may be influenced by metabolites and other (potential) nutraceutical agents.
Currently, only the athletic performance area, including the body-building arena, makes use of dietary supplements targeted towards building muscle. In recent years, this field of use has seen some products sold as dietary supplements that contain ingredients that are in contradiction of the Dietary Supplement Health and Education Act (which governs dietary supplements in the United States). A need for ingredients in dietary supplements that may, in theory, aid muscle mass accretion without the use of banned substances (such as anabolic steroids) exists and was an impetus for the research described herein. However, the resulting data are not limited to this application but has wider reaching implications.
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