Abstract
Exercise is known to create a transient, but potent increase in skeletal muscle expression of potentially anti‐inflammatory myokine interleukin‐6 (IL‐6). This effect may be clinically important in managing chronic inflammatory states. It has previously been proposed that lactic acidosis following exercise promotes this IL‐6 up‐regulation, but the mechanism of this acidosis effect is unknown. Rat skeletal muscle cell line L6‐G8C5 has been used previously to model metabolic effects of acidosis, sensing low pH through the resulting inhibition of amino acid transporter SNAT2(SLC38A2). Use of ionophore ionomycin to model the rise in intracellular Ca2+ concentration occurring in contracting muscle strongly up‐regulates IL‐6 mRNA in L6‐G8C5 myotubes. This study used this model to test the hypothesis that low extracellular pH (7.1) enhances ionomycin‐induced IL‐6 mRNA up‐regulation by inhibiting SNAT2. Incubation of L6‐G8C5 myotubes for 6 h with 0.5 µM ionomycin at control pH (7.4) resulted in a 15‐fold increase in IL‐6 mRNA which was further enhanced (1.74‐fold) at pH 7.1. In contrast low pH had no significant effect on IL‐6 mRNA without ionomycin, nor on the IL‐6 mRNA increase that was induced by cyclic stretch. Even though pH 7.1 halved the transport activity of SNAT2, alternative methods of SNAT2 inhibition (JNK inhibitor SP600125; SNAT2 antagonist MeAIB; or SNAT2 silencing with siRNA) did not mimic the enhancing effect of low pH on IL‐6 mRNA. On the contrary, JNK inhibition blunted the effect of pH 7.1 with ionomycin, but had no effect at pH 7.4. It is concluded that low pH promotes Ca2+/ionomycin–induced up‐regulation of IL‐6 mRNA through a novel SNAT2‐independent JNK‐dependent pH‐sensing pathway not previously described in this skeletal muscle model.
Highlights
In humans, moderate physical exercise is reported to exert significant anti-inflammatory effects,[1] in contrast with the pro-inflammatory effects of intense exercise.[2]
In agreement with the earlier report,[13] treatment of L6- G8C5 rat myotubes for 6 h with ionomycin led to an increase[43] in IL-6 mRNA (41 ± 6 fold increase, p < 0.0001 in Figure 1A and 15 ± 1 fold increase, p < 0.0001 in Figure 1C) which was almost abolished by pharmacological blockade of p38 MAP kinase with the selective inhibitor SB202190 (0.085 ± 0.005 fold decrease, p < 0.0001) (Figure 1A)
Modelling of the metabolic effects of acidosis has previously been performed in this pH-responsive cell line by lowering the pH of the culture medium to 7.1.17–19,21 Here exposure of the cultures to pH 7.1 for 6 h significantly enhanced the stimulatory effect of ionomycin on IL-6 mRNA when compared with control cultures with ionomycin at pH 7.4 (1.74 ± 0.097 fold increase, p < 0.0001; Figure 1C)
Summary
Moderate physical exercise is reported to exert significant anti-inflammatory effects,[1] in contrast with the pro-inflammatory effects of intense exercise.[2]. — which is known as sodium-coupled neutral amino acid transporter 2, SNAT2, SAT2, or ATA2 and will be referred to in the remainder of this paper as SNAT2 It is expressed in skeletal muscle[27] and is the dominant System A transporter of L6-G8C5 cells.[17,18] Inhibition of SNAT2 transport activity by low extracellular pH in these cells leads to depletion of intracellular free amino acids which is sensed by the mTORC1 signalling complex, resulting in impaired protein synthesis.[17] low pH acting through. To investigate (by transport inhibition or by gene silencing of SNAT2) the role of this transporter in mediating pH effects on IL-6 mRNA
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