Ecdysteroids elicit a rapid Ca2+ flux leading to Akt activation and increased protein synthesis in skeletal muscle cells
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 has been well studied, their role in mammals is less understood. Ecdysteroids have been reported to produce a wide range of effects in mammals [1]. One of the most interesting properties of ecdysteroids in mammals is their anabolic effect, behaving similar to anabolic steroids putatively without the androgenic effect. Recently, ecdysteroids were shown to increase muscle fibers in rat [2]. Ecdysteroids have been shown to increase growth and endurance in animals [1], [3] without negative side effects.
Using the murine skeletal muscle cell line C2C12, we have previously shown that treatment with ecdysteroids increases protein synthesis by up to 20% [4]. However, despite evidence showing both in vivo and in vitro anabolic effects of ecdysteroids, their cellular mode of action has not been elucidated. In mammals, there is no known nuclear receptor that is homologous to the ecdysone nuclear receptor (EcR) found in insects, ruling out the possibility that ecdysteroids function through similar pathways in both mammals and insects. Ecdysteroids do not appear to activate the nuclear androgen receptor (AR) since 20-hydroxyecdysone (20HE), the most common ecdysteroid, did not bind to the rat AR [4].
In addition to classical nuclear receptor responses, mammalian steroid hormones structurally related to ecdysteroids have been shown to elicit rapid non-genomic signaling events that mediate cell proliferation and survival. These responses may involve secondary signals such as altered Ca2+ or cAMP [5]. Mammalian steroid hormones increased Ca2+ influx in a variety of cell types including cardiac myocytes [6] and skeletal muscle [7]. Although some of these effects have been attributed to a new role for already identified nuclear receptors, such as the estrogen receptor (ER) and AR, there are data supporting the presence of distinct membrane-bound receptors that interact with these hormones. Estrogen and progesterone G-protein coupled receptors (GPCRs) have already been cloned and characterized and a putative membrane-bound AR has been described [8].In addition to their classical nuclear receptor-mediated genomic response, ecdysteroids also produce many “non-genomic” effects in invertebrates [9], [10]. Although many of these effects may involve the classical nuclear EcR, a putative membrane-bound ecdysone receptor that has been described in silkworms may also be responsible [11]. A membrane-bound GPCR, DopEcR, has been identified in Drosophila which binds ecdysteroids in addition to dopamine [12]. A similar receptor may be responsible for the rapid effects of both androgens and ecdysteroids in mammals.
In this study we used the mouse skeletal muscle cell line, C2C12, to investigate the intracellular responses that may underlie the anabolic effects of ecdysteroids and compared them with the effects of insulin-like growth factor 1 (IGF-1), a well characterized anabolic agent. Specifically, we investigated the ability of 20HE to affect intracellular calcium fluxes. We hypothesized that this might lead to Akt activation via a G-protein coupled receptor–phospholipase c–phosphoinositide-3-kinase (GPCR–PLC–PI3K)-mediated mechanism.
Section snippets
Materials
1,2-Bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Calbiochem (San Diego, CA). Fluo-4 NW was purchased from Invitrogen (Carlsbad, CA). Phospho-Akt and Akt antibodies were purchased from Cell Signaling (Danvers, MA). 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002), Bordetella pertussis
Intracellular Ca2+
Treatment with 1 μM 20HE increased intracellular Ca2+ in C2C12 myotubes within 10 s (Fig. 1A). Intracellular Ca2+ peaked 35 s after treatment, and began to decline after 70 s. The decrease was gradual with some elevation of intracellular Ca2+ still observed after 180 s. This effect was completely abolished when cells were pretreated with 1 μg/ml PTX, 1 h prior to 20HE treatment (Fig. 1B). Ca2+ free media containing the extracellular Ca2+ chelator, 3 mM EGTA, slightly reduced and modified the 20HE
Discussion
20HE produced rapid responses in C2C12 myotubes, including increasing Ca2+ flux in seconds (Fig. 1A) and phosphorylating Akt within 2 h (Fig. 3A and B).
The G-protein inhibitor, PTX completely abolished the 20HE-induced increase in both Ca2+ (Fig. 1A) and protein synthesis (Fig. 8), and significantly reduced Akt activation (Fig. 5), suggesting a G-protein-dependant pathway is involved in both the rapid and long term response. Increased intracellular Ca2+, which is involved in GPCR signaling may
Acknowledgments
Research supported byFogarty International Center of the NIH under U01 TW006674 for the International Cooperative Biodiversity Groups; NIH Center for Dietary Supplements Research on Botanicals and Metabolic Syndrome, grant # 1-P50 AT002776-01; Rutgers University, and Phytomedics Inc. (Jamesburg, NJ, USA);
References (29)
- et al.
20-Hydroxyecdysone increases fiber size in a muscle specific fashion in rat
Phytomedicine
(2008) - et al.
Non-classical actions of testosterone: an update
Trends Endocrinol Metab
(2007) - et al.
Non-genomic ecdysone effects and the invertebrate nuclear steroid hormone receptor EcR--new role for an “old” receptor?
Mol Cell Endocrinol
(2006) - et al.
Nongenomic action of an insect steroid hormone in steroid-induced programmed cell death
Mol Cell Endocrinol
(2007) - et al.
Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes
Cell Calcium
(2000) - et al.
Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells
J Biol Chem
(1999) - et al.
Estradiol binding to cell surface raises cytosolic free calcium in T cells
FEBS Lett
(1998) - et al.
Testosterone induces Ca2+ influx via non-genomic surface receptors in activated T cells
FEBS Lett
(1997) - et al.
Testosterone enhances calcium reabsorption by the kidney
Mol Cell Endocrinol
(2004) - et al.
Impact of testosterone on cardiac L-type calcium channels and Ca2+ sparks: acute actions antagonize chronic effects
Cell Calcium
(2007)
Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells
J Biol Chem
Signal transduction of erythrocytes after specific binging of ecdysterone and cholesterol immobilized on nanodispersed magnetite
J Magn Magn Mater
Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals
J Endocrinol
Anabolic activity of phytoecdysone–ecdysterone isolated from Rhaponticum carthamoides (Willd.) Iljin
Farmakol Toksikol
Cited by (69)
Nutrient weight against sarcopenia: regulation of the IGF-1/PI3K/Akt/FOXO pathway in quinoa metabolites
2021, Current Opinion in PharmacologyDiversity-oriented synthesis through gamma radiolysis: Preparation of unusual ecdysteroid derivatives activating Akt and AMPK in skeletal muscle cells
2021, Bioorganic ChemistryCitation Excerpt :According to the recently proposed canonical mechanism of membrane action of ecdysteroids, the various bioactivities of these compounds may be explained via their activating effect on the Mas receptor, i.e. the G-protein-coupled receptor of angiotensin-(1–7) [51]. This would then lead to a rapid, Ca2+-dependent activation of Akt [14,51], which kinase is therefore a rational choice to evaluate the potential of ecdysteroids for stimulating skeletal muscle protein synthesis [52], and for exerting their protective action on various tissues. Akt activation may also offer a potential strategy to ameliorate insulin resistance [53].
The phytochemical, biological, and medicinal attributes of phytoecdysteroids: An updated review
2021, Acta Pharmaceutica Sinica BCitation Excerpt :Akt is a serine/threonine-kinase which plays multiple key roles in cellular signaling and metabolic processes, such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. Akt activation followed by elevation in intracellular calcium levels led to an increased protein synthesis in a mouse skeletal muscle mytotube cell line C2C12164. Although the mechanism behind PE-mediated activation of Akt is not clear, its downstream effectors represent a major signaling pathway that controls protein turnover and may regulate skeletal muscle activity, which also has been reported for maintaining the aging of muscles163.
The insect molting hormone 20-hydroxyecdysone protects dopaminergic neurons against MPTP-induced neurotoxicity in a mouse model of Parkinson's disease
2020, Free Radical Biology and Medicine