Excessive Endoplasmic Reticulum Stress Correlates with Impaired Mitochondrial Dynamics, Mitophagy and Apoptosis, in Liver and Adipose Tissue, but Not in Muscles in EMS Horses

Abstract

Nowadays, endocrine disorders have become more frequent in both human and veterinary medicine. In horses, reduced physical activity combined with carbohydrate and sugar overload may result in the development of the so-called equine metabolic syndrome (EMS). EMS is characterized by insulin resistance, hyperinsulinemia, elevated blood triglyceride concentrations and usually obesity. Although the phenotypic features of EMS individuals are well known, the molecular mechanism underlying disease development remains elusive. Therefore, in the present study, we analyzed insulin-sensitive tissues, i.e., muscles, liver and adipose tissue in order to evaluate insulin resistance and apoptosis. Furthermore, we assessed mitochondrial dynamics and mitophagy in those tissues, because mitochondrial dysfunction is linked to the development of metabolic syndrome. We established the expression of genes related to insulin resistance, endoplasmic reticulum (ER) stress and mitochondria clearance by mitophagy using RT-PCR and Western blot. Cell ultrastructure was visualized using electron transmission microscopy. The results indicated that adipose tissue and liver of EMS horses were characterized by increased mitochondrial damage and mitophagy followed by triggering of apoptosis as mitophagy fails to restore cellular homeostasis. However, in muscles, apoptosis was reduced, suggesting the existence of a protective mechanism allowing that tissue to maintain homeostasis.

Keywords: apoptosis; autophagy; insulin resistance; metabolic syndrome; mitochondria.

Figure 1

Oxidative stress and cytokines levels in the serum of healthy (CTRL) and EMS horses. Using commercially available, spectrophotometric assays, ROS (A), NO (B) and SOD (C) activity was assessed. The cytokine profile was evaluated with ELISA for IL-10 (D), TNF-α (E) and IL-1β (F). Results expressed as the mean ± standard deviation (S.D.) ** p < 0.01, *** p < 0.001.

Figure 2

Expression of insulin resistance-related genes in muscle, adipose tissue and liver of horses. Using RT-PCR, the expression of IR (A), IRS (B), S6K1 (C), GLUT-4 (D), RBP4 (E) and SREBP1C (F) was investigated. Results expressed as the mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Evaluation of apoptosis in muscle, adipose tissue and liver of healthy (CTRL) and EMS individuals. Using RT-PCR, we evaluated the expression of p53 (A), p21 (B), BAX (C), Bcl-2 (D) and the Bcl-2/BAX ratio (E). Moreover, the amount of caspase-3 in samples was established by Western blot analysis (F). Results expressed as the mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Investigation of ER stress in tissues from healthy and EMS horses. Using RT-PCR, the expression of CHOP (A), PERK (B) and IL-13 (C) was examined. Furthermore, TEM images allowed for visualization of cellular ultrastructure and detection of organelles’ impairment (D). Abbreviations: ER, endoplasmic reticulum; eER, enlarged endoplasmic reticulum; LD, lipid droplets; PN, pyknotic nucleus. Results expressed as the mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Evaluation of autophagy in muscle, adipose tissue and liver from healthy and EMS individuals. Using RT-PCR, the expression of mTOR (A), PI3K (B), AKT (C), Beclin-3 (D), LC3 (E), the ratio of Beclin/LAMP2 (F) and LAMP2 (G) was investigated. Using immunofluorescence, intracellular localization of LAMP2 in samples was examined (H). TEM analysis allowed for visualization of autophagosomes’ formation (indicated with red arrows (I)). LD, lipid droplets; AP, autophagosomes. Results expressed as the mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: confocal 20 µm, transmission electron microscope (TEM) 200 nm.

Figure 5

Evaluation of autophagy in muscle, adipose tissue and liver from healthy and EMS individuals. Using RT-PCR, the expression of mTOR (A), PI3K (B), AKT (C), Beclin-3 (D), LC3 (E), the ratio of Beclin/LAMP2 (F) and LAMP2 (G) was investigated. Using immunofluorescence, intracellular localization of LAMP2 in samples was examined (H). TEM analysis allowed for visualization of autophagosomes’ formation (indicated with red arrows (I)). LD, lipid droplets; AP, autophagosomes. Results expressed as the mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: confocal 20 µm, transmission electron microscope (TEM) 200 nm.

Figure 6

Evaluation of mitophagy and mitochondrial dynamics in tissues from healthy and EMS-diagnosed horses. Using RT-PCR, the expression of PINK (A), PARKIN (B), MNF (C) and FIS (D) was investigated. PARKIN amount was also examined by Western blot (E) and immunofluorescence staining (F). TEM imaging (F) allowed for visualization of mitophagosomes’ formation (indicated with yellow arrows) and mitochondria morphology impairment (indicated with red arrows). MP, mitophagosomes; IM, impaired mitochondria. Results expressed as the mean ± S.D. ** p < 0.01, *** p < 0.001. Scale bars: confocal 20 µm, TEM 100 nm.

Figure 6

Evaluation of mitophagy and mitochondrial dynamics in tissues from healthy and EMS-diagnosed horses. Using RT-PCR, the expression of PINK (A), PARKIN (B), MNF (C) and FIS (D) was investigated. PARKIN amount was also examined by Western blot (E) and immunofluorescence staining (F). TEM imaging (F) allowed for visualization of mitophagosomes’ formation (indicated with yellow arrows) and mitochondria morphology impairment (indicated with red arrows). MP, mitophagosomes; IM, impaired mitochondria. Results expressed as the mean ± S.D. ** p < 0.01, *** p < 0.001. Scale bars: confocal 20 µm, TEM 100 nm.