Delaying of insulin signal transduction in skeletal muscle cells by selenium compounds

https://doi.org/10.1016/j.jinorgbio.2011.03.010Get rights and content

Abstract

Supranutritional selenium (Se) intake and high serum Se levels have been associated epidemiologically with increased risk for type 2 diabetes, suggesting adverse effects of dietary Se compounds and/or antioxidant selenoenzymes on the sensitivity of target tissues for insulin. Here, we compared the capability of inorganic (sodium selenite and sodium selenate) and organic (selenomethionine and methylseleninic acid (MSeA)) Se compounds to interfere with insulin signaling in rat L6 myotubes, differentiated skeletal muscle cells. When applied at doses of 1 μM, only selenite and MSeA were capable of delaying insulin-induced phosphorylation of protein kinase B (Akt) and attenuating insulin-induced phosphorylation of forkhead box class O transcription factors FoxO1a and FoxO3. Insulin-stimulated glucose uptake was lowered by selenite and MSeA as well. Even though all tested Se compounds strongly stimulated expression/activity of the cellular selenoproteins glutathione peroxidase 1 and selenoprotein W, selenite and MSeA were the most efficiently utilized Se donors. Moreover, at doses of 1 μM, only selenite and MSeA had a significant inhibitory effect on generation of intracellular reactive oxygen species (ROS). These results suggest that the Se(IV) compounds selenite and MSeA may impair the insulin sensitivity of myocytes by influencing cellular redox homeostasis.

Graphical abstract

The selenium(IV) compounds selenite and methylseleninic acid impair the insulin sensitivity of skeletal muscle cells by attenuating intracellular ROS production.

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Introduction

Among the trace elements essential for human health, selenium (Se) stands out for its unique biochemistry, its antioxidant capacity and its narrow therapeutic window. Selenocysteine, the selenium analog of cysteine, is co-translationally incorporated into 25 human selenoproteins [1]. Glutathione peroxidases (GPx), selenoprotein P (SeP) and thioredoxin reductases are the most prominent and ubiquitously expressed selenoproteins, contributing to degradation of reactive oxygen species (ROS) and regulation of cellular redox homeostasis [2]. Se is unequally distributed in the human body: Se levels in tissues of German adults have been reported to range from 110 ng Se/g in skeletal muscle and brain to 770 ng Se/g in kidney [3]. Nevertheless, skeletal muscle contains 50% of whole body Se because of its high mass [3]. Depending on the content and the chemical form of Se in the diet, Se concentrations in human plasma can vary considerably, ranging from 20 μg/L (0.25 μM) to 95 μg/L (1.2 μM) in Chinese subjects and from 79 μg/L (1.0 μM) to 147 μg/L (1.86 μM) in US-Americans [4]. Se is present in human plasma in the form of two selenoproteins, SeP and GPx3, as well as in a non-selenoprotein Se pool bound to albumin [5]. A daily intake of 30–85 μg Se is recommended for adequate Se supply, based on dietary requirements for saturation of plasma GPx3 activity, whereas the optimization of plasma SeP levels requires a higher intake of 90–100 μg Se/day [6], [7], [8]. Assumed health benefits, in particular regarding the prevention of oxidative stress-related illnesses such as cancer or neurodegenerative diseases [2], [6], prompted many individuals to consume substantial amounts of Se-enriched dietary supplements. However, its narrow therapeutic window makes selenium a “double-edged sword”: supranutritional Se intake may trigger adverse health effects even below the “tolerable upper intake level”, currently set at 400–450 μg Se/day for adults [6].

An ongoing discussion questioning usefulness and safety of dietary Se supplements has arisen: participants of two recent intervention trials did not benefit from Se supplementation, either in terms of improved cardiovascular function or in terms of tumor prevention [9], [10]. It is noteworthy that the participants had high baseline plasma Se levels, pointing to already saturated expression/activity of selenoproteins at the beginning of the studies. A secondary analysis of data from the Nutritional Prevention of Cancer (NPC) trial revealed that subjects, who had received over 12 years a daily dose of 200 μg Se in the form of high Se-yeast, were more likely to develop type 2 diabetes than those assigned to placebo [11]. The highest diabetes risk was noticed in the subgroup of Se-supplemented individuals with highest initial plasma Se levels (> 122 μg/L) [11]. An association between high plasma Se levels and increased prevalence of type 2 diabetes, hyperglycemia and dyslipidemia is particularly evident in the selenium-replete US-American population, but similar results have also been reported in several European studies [11], [12], [13], [14]. Apparently, consumption of Se supplements at high doses may disturb the metabolism of carbohydrates and lipids, due to adverse effects of dietary Se compounds and/or abundantly expressed selenoenzymes on the insulin sensitivity of major insulin target tissues and/or on the regulation of pancreatic insulin biosynthesis [13], [14].

The idea to regard high Se intake as a risk factor for type 2 diabetes may appear paradoxical, bearing in mind existing evidence for insulin-mimetic and anti-diabetic actions of inorganic Se compounds such as sodium selenate and sodium selenite [13], [14], [15], [16]. Moreover, the antioxidant ROS-detoxifying capacity of selenoenzymes suggests that Se may protect against late complications of diabetes caused by chronic oxidative stress [17]. On the other hand, abundant expression/activity of selenoproteins may interfere with insulin signal transduction and impair metabolic pathways related to insulin signaling: transgenic mice overexpressing the selenoenzyme GPx1 developed, at older age, a type 2 diabetes-like phenotype characterized by insulin resistance, hyperglycemia, hyperinsulinemia and obesity [18], whereas improved insulin sensitivity was observed in skeletal muscle of GPx1 knock-out mice [19]. In the skeletal muscle of transgenic mice with deficient biosynthesis of selenoproteins, site-specific phosphorylation of two components of the insulin signaling cascade, protein kinase B (Akt) and p70 ribosomal S6 kinase (p70 S6 kinase), was increased [20]. As plasma GPx activity in humans is saturated at a daily Se intake well below the doses associated with increased diabetes risk [6], [7], [8], [11], [12], [13], it appears unlikely that only glutathione peroxidases mediate the pro-diabetic effects of high Se intake. In addition to GPx, selenoprotein P and/or Se compounds of the non-selenoprotein Se pool in plasma might impair insulin-induced signaling pathways. Indeed, selenoprotein P has been recently demonstrated to induce insulin resistance in hepatocytes and myocytes [21]. In regard to the inconsistent data on pro- and anti-diabetic effects of Se, the present study was undertaken to compare the influence of inorganic and organic Se compounds on the insulin sensitivity of skeletal muscle cells, a major target of insulin. l-selenomethionine (SeMet), sodium selenite and sodium selenate are sources of Se in foods and/or dietary supplements [6]. Methylseleninic acid (MSeA) has been used for cancer prevention [22].

Section snippets

Reagents and antibodies

Selenium compounds and insulin were purchased from Sigma (Taufkirchen, Germany). The primary antibodies used in this study were: anti-β-actin, anti-pan-Akt, anti-phospho-Akt(Ser473), anti-phospho-FoxO1a(Thr24)/FoxO3(Thr32) (Cell Signaling Technology; Beverly, MA) and anti-selenoprotein W (Rockland; Gilbertsville, PA). The secondary HRP-coupled anti-rabbit IgG antibody was obtained from Dianova (Hamburg, Germany). Reagents for SDS-PAGE were from Roth (Karlsruhe, Germany). PCR primers were

Utilization of Se compounds for the synthesis of selenoproteins in L6 myotubes

The experimental protocol for differentiation of L6 rat myoblasts into myotubes and their supplementation with Se compounds was established according to previous studies in the L8 rat skeletal muscle cell line [26], [27]. We examined the expression of the muscle intermediate filament protein desmin as marker of myocyte differentiation [28]. Desmin mRNA levels increased steadily over the course of differentiation, being 18-folds higher in myotubes (day 6 of differentiation) compared to

Discussion

The results presented here demonstrate the capability of two selenium compounds, sodium selenite and methylseleninic acid (MSeA), to counteract insulin-induced signaling in myocytes (Fig. 7). In humans, skeletal muscle accounts for about 75% of insulin-dependent glucose uptake and storage in the fed state, making insulin resistance of this major target tissue a prominent feature of type 2 diabetes [32]. Insulin sensitivity is influenced by cellular redox homeostasis. Intracellular ROS such as

Abbreviations

    Akt

    protein kinase B

    DCF

    2′,7′-dichlorofluorescein

    DMEM

    Dulbecco's modified Eagle's medium

    FoxO

    forkhead box protein class O

    Glut4

    glucose transporter 4

    GPx

    glutathione peroxidase

    H2O2

    hydrogen peroxide

    HPRT1

    hypoxanthine phosphoribosyltransferase 1

    IGF-I

    insulin-like growth factor I

    MSeA

    methylseleninic acid

    NHANES III

    Third National Health and Nutrition Examination Survey

    NPC

    Nutritional Prevention of Cancer Trial

    p70 S6 kinase

    p70 ribosomal S6 kinase

    ROS

    reactive oxygen species

    RT-PCR

    reverse transcription polymerase

Acknowledgments

This study was supported by Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany (STE 1782/2-1 and Sonderforschungsbereich 575/B4). H. Sies is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD. We thank Dr. S. Schinner and Dr. M. Ouwens for the helpful discussions, and A. Borchardt for the excellent technical assistance.

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