Elsevier

Steroids

Volume 75, Issue 10, October 2010, Pages 632-637
Steroids

Ecdysteroids elicit a rapid Ca2+ flux leading to Akt activation and increased protein synthesis in skeletal muscle cells

https://doi.org/10.1016/j.steroids.2010.03.008Get rights and content

Abstract

Phytoecdysteroids, structurally similar to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. In skeletal muscle cells, phytoecdysteroids increase protein synthesis. In this study we show that in a mouse skeletal muscle cell line, C2C12, 20-hydroxyecdysone (20HE), a common phytoecdysteroid in both insects and plants, elicited a rapid elevation in intracellular calcium, followed by sustained Akt activation and increased protein synthesis. The effect was inhibited by a G-protein coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, and a phosphoinositide kinase-3 (PI3K) inhibitor.

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);

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