Exhaustive acute exercise-induced ER stress is attenuated in IL-6-knockout mice

in Journal of Endocrinology
Correspondence should be addressed to A S R da Silva: adelinosanchez@usp.br

The endoplasmic reticulum (ER) stress and inflammation relationship occurs at different levels and is essential for the adequate homeostatic function of cellular systems, becoming harmful when chronically engaged. Intense physical exercise enhances serum levels of interleukin 6 (IL-6). In response to a chronic exhaustive physical exercise protocol, our research group verified an increase of the IL-6 concentration and ER stress proteins in extensor digitorium longus (EDL) and soleus. Based on these results, we hypothesized that IL-6-knockout mice would demonstrate a lower modulation in the ER stress proteins compared to the wild-type mice. To clarify the relationship between exercise-induced IL-6 increased and ER stress, we studied the effects of an acute exhaustive physical exercise protocol on the levels of ER stress proteins in the skeletal muscles of IL-6-knockout (KO) mice. The WT group displayed a higher exhaustion time compared to the IL-6 KO group. After 1 h of the acute exercise protocol, the serum levels of IL-6 and IL-10 were enhanced in the WT group. Independent of the experimental group, the CHOP and cleaved caspase 12/total caspase 12 ratio in EDL as well as ATF6 and CHOP in soleus were sensitive to the acute exercise protocol. Compared to the WT group, the oscillation patterns over time of BiP in EDL and soleus as well as of peIF2-alpha/eIF2-alpha ratio in soleus were attenuated for the IL-6 KO group. In conclusion, IL-6 seems to be related with the ER stress homeostasis, once knockout mice presented attenuation of BiP in EDL and soleus as well as of pEiF2-alpha/EiF2-alpha ratio in soleus after the acute exhaustive physical exercise protocol.

 

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    Western blot calibration method. Firstly, the Western blots for the same protein at the basal time, 1h, and 3h were performed in the same tank at the same time. Nevertheless, to compare the results between these Western blots, we utilized a calibration method as follow described: At the end of each Western blot membrane, a sample was inserted and used as an internal control. The amount of protein used for this internal control was always the same. Therefore, we assumed the signals obtained after membrane revelations would be similar. When any variation occurred, we considered as an inherent variability among Western blots run in the same tank at the same time. Based on this possibility, we standardized that all experimental samples should be corrected by a calibration factor, which was obtained by the ratio between the internal controls of the different membranes.

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    Internal control test. (A) Western blot test using the internal control at the end of each Western blot membrane. (B) Graph (arbitrary units).

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    Exhaustion time in minutes for the WT and KO groups (A). Gastrocnemius glycogen concentrations for the WT and KO groups at the basal time, 1 h and 3 h after the acute exhaustive physical exercise protocol (B). Data correspond to means ± s.e. of n = 30 mice. WT, wild-type animals (n = 15). KO, IL-6-knockout animals (n = 15). *P < 0.05 vs WT group.

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    Serum levels (pg/mL) of IL-1beta (A), IL-6 (B), IL-10 (C), IL-15 (D) and TNF-alpha (E), which were measured at the basal time, 1 h and 3 h after the acute exhaustive exercise protocol for the experimental groups. Data correspond to means ± s.e. of n = 30 mice. WT, wild-type animals (n = 15). KO, IL-6 knockout animals (n = 15). *P < 0.05 vs WT group for the same time; +P < 0.05 vs WT group at the basal time; ++P < 0.05 vs KO group at the basal time; #P < 0.05 vs KO group at 1 h after the acute exhaustive exercise protocol.

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    Protein levels (arbitrary units) in EDL of beta-actin, BiP and ATF6 (A, B, C, D, E and F), which were measured at the basal time, 1 h and 3 h after the acute exhaustive exercise protocol for the experimental groups. Data correspond to means ± s.e. of n = 30 mice. WT, wild-type animals (n = 15). KO, IL-6-knockout animals (n = 15). *P < 0.05 vs WT group at the same time.

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    Protein levels (arbitrary units) in EDL of beta-actin, peIF2-alpha and eIF2-alpha (A, B and C), which were measured at the basal time, 1 h and 3 h after the acute exhaustive exercise protocol for the experimental groups. Data correspond to means ± s.e. of n = 30 mice. WT, wild-type animals (n = 15). KO, IL-6-knockout animals (n = 15).

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    Protein levels (arbitrary units) in EDL of GAPDH, CHOP, alpha-tubulin, beta-actin, total caspase 12 and cleaved caspase 12 (A, B, C, D, E and F), which were measured at the basal time, 1 h and 3 h after the acute exhaustive exercise protocol for the experimental groups. Data correspond to means ± s.e. of n = 30 mice. WT, wild-type animals (n = 15). KO, IL-6 knockout animals (n = 15). +P < 0.05 vs WT group at the basal time; ++P < 0.05 vs KO group at the basal time; #P < 0.05 vs KO group at 1 h after the acute exhaustive exercise protocol.

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    Protein levels (arbitrary units) in soleus of alpha-tubulin, BiP and ATF6 (A, B, C, D, E and F), which were measured at the basal time, 1 h and 3 h after the acute exhaustive exercise protocol for the experimental groups. Data correspond to means ± s.e. of n = 30 mice. WT, wild-type animals (n = 15). KO, IL-6-knockout animals (n = 15). *P < 0.05 vs WT group at the same time; +P < 0.05 vs WT group at the basal time; ++P < 0.05 vs KO group at the basal time; #P < 0.05 vs KO group at 1 h after the acute exhaustive exercise protocol.

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    Protein levels (arbitrary units) in soleus of alpha-tubulin, peIF2-alpha, eIF2-alpha and GAPDH, (A, B and C), which were measured at the basal time, 1 h and 3 h after the acute exhaustive exercise protocol for the experimental groups. Data correspond to means ± s.e. of n = 30 mice. WT, wild-type animals (n = 15). KO, IL-6-knockout animals (n = 15). +P < 0.05 vs WT group at the basal time.

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    Protein levels (arbitrary units) in soleus of GAPDH, CHOP, alpha-tubulin, total caspase 12 and cleaved caspase 12 (A, B, C, D, E and F), which were measured at the basal time, 1 h and 3 h after the acute exhaustive exercise protocol for the experimental groups. Data correspond to means ± s.e. of n = 30 mice. WT, wild-type animals (n = 15). KO, IL-6 knockout animals (n = 15). ++P < 0.05 vs KO group at the basal time; #P < 0.05 vs KO group at 1 h after the acute exhaustive exercise protocol.

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    Schematic figure summarizing the findings of the present investigation.

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