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NEW STUDY ON ECDYSTEROIDS!
J. Agric. Food Chem., 56 (10), 3532?3537, 2008. 10.1021/jf073059z
Web Release Date: April 30, 2008
Copyright ? 2008 American Chemical Society
Phytoecdysteroids Increase Protein Synthesis in Skeletal Muscle Cells
Jonathan Gorelick-Feldman,*? David MacLean,? Nebojsa Ilic,? Alexander Poulev,? Mary Ann Lila,# Diana Cheng,# and Ilya Raskin?
Biotech Center, Cook College, Rutgers University, 59 Dudley Road, New Brunswick, New Jersey 08901;Hallett Center for Diabetes and Endocrinology, Brown Medical School, Providence, Rhode Island 02912; and Department of Natural Resources and Environmental Sciences, University ofIllinois, Urbana, Illinois 61801
Abstract:
Phytoecdysteroids, which are structurally similar or identical to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. To study the mechanism of action of phytoecdysteroids in mammalian tissue, an in vitro cellular assay of protein synthesis was developed. In C2C12 murine myotubes and human primary myotubes, phytoecdysteroids increased protein synthesis by up to 20%. In vivo, ecdysteroids increased rat grip strength. Ecdysteroid-containing plant extracts produced similar results. The effect was inhibited by a phosphoinositide kinase-3 inhibitor, which suggests a PI3K-mediated mechanism.
Introduction
Ecdysteroids, polyhydroxylated ketosteroids with long carbon side chains, are produced primarily in insects and plants. Although the role of ecdysteroids as insect hormones and their involvement in development and the molting process have been well studied, their role in plants is less obvious. Ecdysteroids found in plants, called phytoecdysteroids, do not elicit any of the classical plant hormone responses; however, they do elicit weak gibberellin-like activity in rice, as well as affect differentiation in alfalfa embryos (1). It has been suggested that plants utilize ecdysteroids as a chemical defense against insect herbivory by disrupting the insect hormonal balance and molting process (2, 3).
Ecdysteroids have also been reported to have effects in mammals, including lowering cholesterol levels (4) and blood glucose (5). They have immunomodulating (6), antiarrythymic (7), and hepatoprotective effects [(8); for a review see refs (9) and (10)]. Despite all of these effects, no receptor homologous to the insect ecdysone receptor has been identified in vertebrates. In fact, the transfected ecdysone receptor has been utilized for gene switch systems (9).
Anabolic effect is another reported property of ecdysteroids in vertebrates. Ecdysteroids have been shown to increase growth in a wide variety of animals including mice (11, 12), rats (13), sheep (14), pigs (15), and quail (16). Observed anabolic effects of these compounds are increased physical performance without training, as demonstrated using the forced swim test with rats, and increased synthesis of myofibrillar proteins in both the soleus and extensor digitorum longus (17). Increased growth and protein content were also observed in ecdysteroid-treated mouse liver and kidneys (18).
Plants are natural sources of ecdysteroids. Although most plants do not contain measurable amounts of ecdysteroids, some plants produce high levels of these compounds. Ajuga turkestanica, an herb from the basil family native to Uzbekistan, contains high levels of the C-11 hydroxylated turkesterone, one of the more active ecdysteroids (19). The high concentration of this potent ecdysteroid makes A. turkestanica a potentially useful medicinal plant. Edible plants, such as Spinacia oleracea (spinach), also contain considerable amounts of ecdysteroids, such as 20-hydroxyecdysone (20HE) (2), one of the most common plant-derived ecdysteroids. In addition to its use as a food crop, spinach may also have potential therapeutic qualities.
Despite the data showing various in vivo anabolic effects of ecdysteroids, the mechanisms of their cellular mode of action have not been elucidated. One of the barriers has been the lack of a simple in vitro assay to quantify the anabolic effect of ecdysteroids on skeletal muscle. In this study, we developed a cell culture-based method for analyzing the effects of ecdysteroids and report a significant increase in the protein synthesis of muscle cells following ecdysteroid treatment. The model was further used to identify factors that either enhanced or abrogated this anabolic effect. In support of earlier literature, we confirmed that this in vitro effect was translated in vivo as demonstrated by increased grip strength in rats treated with 20HE or with ecdysteroid-containing plant extracts.
Materials and Methods
Materials. 20HE, polypodine B, and ponesterone were purchased from Scitech (Praha, Czech Republic). Methandrostenolone, a synthetic anabolic steroid, was purchased from Steraloids (Newport, RI). Pure turkesterone was a gift from the Tashkent Institute of Cardiology (Uzbekistan). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Plant Extraction. A. turkestanica was collected in Uzbekistan, and voucher specimens were cataloged in the Rutgers Herbarium. The dried aerial portion was extracted in 95% ethanol. On the basis of previous phytoecdysteroid extraction protocols (16), the ethanolic extract was partitioned with butanol, and the butanolic phase was dried and used for testing.
Locally grown dried spinach powder (S. oleracea) was extracted in 95% ethanol for 24 h. After the removal of ethanol, the extract was resuspended in water and partitioned with heptane. The organic phase was removed, and the water phase was partitioned with butanol. The butanolic phase was dried and used for testing.
The ecdysteroid content of plant extracts was determined using (+)ESI LC-MS. A standard curve was generated using increasing amounts of purified turkesterone or 20HE. The typical fragmentation ions of ecdysteroids [(M + H) + (M − H2O + H) + (M − 2H2O + H) + (M − 3H2O + H)] were merged to produce chromatograms for each compound. The fragmentation patterns and retention times of the plant extracts were compared with those of standards to quantify the amount of turkesterone and 20HE present.
Cell Culture. A mouse skeletal muscle cell line, C2C12 (ATCC CRL-1772), was maintained according to the method of Montgomery et al. (20). Between passages 3 and 10, cells were seeded at a density of 105 cells/cm2 onto 24-well tissue culture plates. The cells were grown in low-glucose Dulbecco?s Modified Eagle?s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 6 mM glutamine, 1 mM pyruvate, 100 units/mL penicillin, and 100 ?g/mL streptomycin (Gibco, Grand Island, NY). Cells were grown for 48 h in 5% CO2 at 37 ?C. After cells reached 80% confluency, the medium was replaced with differentiation medium (DMEM with 2% horse serum). After 5 days, the myoblasts had fused into multinucleated myotubes.
Primary human skeletal muscle cells (a gift from Dr. William Cefalu of the Pennington Biomedical Research Center, Baton Rouge, LA) were seeded at a density of 105 cells/cm2 onto 24-well tissue culture plates. The cells were grown in DMEM supplemented with 10% FBS and a SingleQuot Kit (Lonza, Portsmouth, NH) containing epidermal growth factor, insulin, bovine serum albumin (BSA), fetuin, dexamethasone, and gentamicin/amphotericin-B. Cells were grown for 96 h in 5% CO2 at 37 ?C until they reached 80% confluency, and the medium was replaced with differentiation medium (DMEM with 2% horse serum). After 18 days, myoblasts fused into multinucleated myotubes.
Cell Treatment. For the ecdysteroid dose response, C2C12 cells were washed with serum-free DMEM and treated with increasing concentrations of 20HE, turkesterone, ponesterone, polypodine B, methandrostenolone, or the vehicle, 0.1% ethanol, four wells per treatment. Compounds were added to serum-free medium containing 5 ?Ci/mL [3H]leucine. Cells were incubated for 4 h before protein measurement. For the 20HE time course study, C2C12 cells were treated with 1 ?M 20HE for 0.5−24 h in serum-free medium containing 5 ?Ci/mL [3H]leucine before protein measurement. For the plant extract study, C2C12 cells were treated with increasing concentrations of spinach extract, A. turkestanica extract, or vehicle for 4 h during the radiolabeled exposure period before protein measurement. For the inhibitor study, C2C12 cells were pretreated with either 10 ?M of the phosphoinositide kinase-3 (PI3K) inhibitor, LY294002, or vehicle for 30 min. The medium was changed, and the cells were incubated with either 1 ?M 20HE, 100 ng/mL insulin-like growth factor-1 (IGF-1), or vehicle for 4 h before protein measurement. For the 24 h 20HE dose response in human muscle cells, the cells were washed and treated with either 1 ?M 20HE or vehicle in serum-free leucine containing medium supplemented with 5 ?Ci/mL [3H]leucine. Cells were then incubated for 24 h before protein measurement.
Protein Synthesis Assay. Protein synthesis was determined by measuring the incorporation of the tritiated amino acid, leucine (20). Briefly, following treatment, cells were washed with cold phosphate-buffered saline (PBS), followed by the addition of 5% trichloroacetic acid (TCA) to precipitate protein. After 30 min at 4 ?C, the TCA was removed and the precipitate was dissolved in 0.5 M NaOH (500 ?L). The dissolved precipitate (400 ?L) was added to scintillation vials with 5 mL of scintillation fluid (Ready Safe, Beckman Coulter, Fullerton, CA). Decays per minute (DPM) were measured in a liquid scintillation counter (LS 6500, Beckman Coulter). Total protein was quantified using the bicinchoninic acid (BCA) method following the manufacturer?s instructions (Pierce, Rockford, IL). The data were expressed as DPM per milligram of total protein. Each experiment was performed in triplicate. The results were expressed as mean SEM. Statistical significance was determined using Student?s t test (p < 0.05).[/QUOTE]
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