Taurine Enhances Iron-Related Proteins and Reduces Lipid Peroxidation in Differentiated C2C12 Myotubes

Abstract

Taurine is a nonproteinogenic amino sulfonic acid in mammals. Interestingly, skeletal muscle is unable to synthesize taurine endogenously, and the processing of muscular taurine changes throughout ageing and under specific pathophysiological conditions, such as muscular dystrophy. Ageing and disease are also associated with altered iron metabolism, especially when there is an excess of labile iron. The present study addresses the question of whether taurine connects cytoprotective effects and redox homeostasis in a previously unknown iron-dependent manner. Using cultured differentiated C2C12 myotubes, the impact of taurine on markers of lipid peroxidation, redox-sensitive enzymes and iron-related proteins was studied. Significant increases in the heme protein myoglobin and the iron storage protein ferritin were observed in response to taurine treatment. Taurine supplementation reduced lipid peroxidation and BODIPY oxidation by ~60 and 25%, respectively. Furthermore, the mRNA levels of redox-sensitive heme oxygenase (Hmox1), catalase (Cat) and glutamate-cysteine ligase (Gclc) and the total cellular glutathione content were lower in taurine-supplemented cells than they were in the control cells. We suggest that taurine may inhibit the initiation and propagation of lipid peroxidation by lowering basal levels of cellular stress, perhaps through reduction of the cellular labile iron pool.

Keywords: BODIPY; glutathione; labile iron pool; myoglobin; skeletal muscle.

Scheme 1

Chemical structure of taurine showing its two functional groups, the amino group and the sulfonic acid group. Taurine is zwitterionic at an isoelectric point of 5.12 [12]. The molecular weight of taurine is 125.15 g/mol and the molecular formula is C2H7NO3S [13].

Figure 1

Taurine (TAUR) was not cytotoxic at concentrations of up to 100 mmol/L (a) Cell viability after 24 h was determined by the uptake of neutral red by taurine-supplemented (1–250 mmol/L) myotubes and untreated control (CON) myotubes. Ethanol (10%) was used as the positive control to induce cytotoxicity. The data are shown as the means + SEM (n = 6–9). (b) Compared to CON, TAUR decreased the mRNA levels of the taurine transporter (TAUT), but TAUT protein levels remained unchanged. TAUT protein levels were quantified densitometrically and normalized according to total protein per lane. A representative section of the target bands and corresponding total protein bands visualized by UV are shown. (c) Optical microscopy images (200×) of differentiated myotubes show morphological changes and cell fragmentation induced by treatment with EtOH and the highest taurine concentration (250 mmol/L), whereas cells incubated with 5 mmol/L taurine did not differ from CON cells. The data are shown as the means + SEM (n = 3 for taurine and TAUT analysis; n = 6 for Taut mRNA experiments). * Indicates significant differences compared to CON; p < 0.05.

Figure 2

Taurine (TAUR) reduced markers for lipid peroxidation (LPO) and cellular redox homeostasis. (a) TAUR reduced the level of secondary LPO products measured as thiobarbituric acid reactive substances (TBARS). (b) To examine LPO under basal and stress conditions, myotubes were treated with the fluorescent dye C11 (undecanoic acid)-BODIPY 581/591, and then DPBS buffer was added in the absence or presence of 2 µmol/L iron(II) sulfate (Fe²⁺) and 80 µmol/L cumene hydroperoxide (CumOOH). TAUR counteracted CumOOH-induced lipid peroxidation. BODIPY oxidation was determined by the ratio of oxidized to total BODIPY (oxidized + reduced) measured as fluorescence intensity. (c) The mRNA levels of heme oxygenase 1 (Hmox1), catalase (Cat) and glutamate-cysteine ligase, catalytic subunit (Gclc) were downregulated after 6 h of treatment with TAUR. (d) Catalase activity was significantly downregulated due to 24 h of incubation with TAUR compared to CON. (e) TAUR decreased the cellular content of total glutathione (tGSH = GSH + GSSG) under basal conditions. The data are shown as the means + SEM (n ≥ 6). * Indicates significant differences compared with CON; p < 0.05. Glutathione (GSH); glutathione disulfide (GSSG).

Figure 3

Taurine enhanced ferritin and myoglobin protein levels. (a) The mean protein levels of ferritin light chain (FTL), cytochrome c (CYTC) and myoglobin (MB) were increased after taurine (TAUR) supplementation. The levels in the untreated control (CON) were set to 1 for each individual experiment, and the results of TAUR supplementation are given relative to those of CON. Target band intensity was densitometrically analyzed and normalized by total protein load per lane, as visualized in a stain-free UV image. Representative pictures of the target bands (FTL, FTH, CYTC and MB) and the corresponding total protein bands are shown. (b) The mRNA levels of iron-sensitive genes Ftl, Fth and Tfr were quantified in response to treatment with TAUR and iron (II) sulfate (Fe²⁺). Fe²⁺, which was used as a positive control, significantly enhanced Ftl and decreased Tfr mRNA levels compared to those of untreated cells. The mRNA levels of Fth were not affected by Fe²⁺ or taurine. (c) A representative picture of target bands indicating the subunits of mitochondrial oxidative phosphorylation (OXPHOS) complexes CI (NADH dehydrogenese), CII (succinate dehydrogenase), CIII (ubiquinol-cytochrome c reductase), CIV (cytochrome c oxidase) and CV (ATP-Synthase) for CON and TAUR and the corresponding total protein load are shown. (d) The relative mRNA levels of NFU1 iron-sulfur cluster scaffold (Nfu1), BolA FAMILY MEMBER 3 (Bola3) and glutaredoxin 5 (Glxr5) were not affected, while nucleotide binding protein like (Nubpl) mRNA was upregulated in response to taurine supplementation. The data are shown as the means + SD (n = 3) for the protein analysis and means + SEM (n = 6) for mRNA results. * Indicates a significant difference; p < 0.05.