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Protective effects of adiponectin receptor agonists against equine lamellar endoplasmic reticulum stress
Marine A Barnabé1, Jonathan Elliott1, Nicola J Menzies-Gow2
1Department of Comparative Biomedical Sciences, Royal Veterinary College, Hertfordshire, UK.
Background:
Lamellar endoplasmic reticulum (ER) stress occurs in hyperinsulinemia-associated and sepsis-related laminitis. Adiponectin is associated with reduced laminitis risk and inhibits ER stress in other species.
Objectives:
To induce ER stress in ex vivo equine lamellar cells using pharmacological agents. To determine whether co-treatment with adiponectin receptor agonists (ARAs) reduces ER stress.
Study Design:
In vitro.
Methods:
Lamellar cells from n = 4-5 horses were treated with tunicamycin or thapsigargin (10-1000 nM) for 2-24 h. ER-stressed cells were co-treated with full-length adiponectin (FLA; 30-3000 ng/mL), globular adiponectin (gA; 30-3000 ng/mL), AdipoRon (2-50 μM), or AICAR (1 mM) for 24 h. Expression of binding immunological protein (BIP) and spliced X-box binding protein (sXBP1; two ER stress markers), ribosomal protein 6 (RPS6), and interleukin-6 and -8 was determined using qRT-PCR and western blotting.
Results:
BIP expression increased in a concentration- and time-dependent manner and was highest after 24 h treatment with 1000 nM tunicamycin (mRNA: mean [95% CI] log2 fold-change [FC] = 4.8 [3.7-5.9]; p < 0.001; protein: log2FC = 3.3 [2.7-4.0]; p < 0.001). XBP1 splicing increased from 6% [4-8] (control) to a maximum of 89% [86-93] (p < 0.001). Thapsigargin induced similar responses. Interleukin-6 (2 h; maximum log2FC = 3.1 [0.7-5.6]; p = 0.03) and interleukin-8 (24 h; maximum log2FC = 5.4 [2.6-8.2]; p = 0.04) increased with both stressors. ER stress-induced BIP protein expression was decreased by AICAR (log2FC = -2.0 [-5.1 to -1.1], p = 0.04) and AdipoRon (maximum log2FC = -1.8 [-2.1 to -1.5]; p = 0.001; tunicamycin), and by FLA (log2FC = -1.3 [-1.8 to -0.7]; p = 0.002), and gA (log2FC = -1.2 [-1.9 to -0.5]; p = 0.004; thapsigargin). Tunicamycin-induced XBP1 splicing decreased with FLA and AdipoRon (maximum change = -11%; p = 0.01), and with all ARAs after thapsigargin treatment (maximum change = -46%; p < 0.001). Total RPS6 expression was decreased by gA, AdipoRon, and AICAR (maximum log2FC = -0.9; p < 0.001; tunicamycin). AdipoRon reduced RPS6 phosphorylation (log2FC = -1.4 [-3.6 to -0.7]; p = 0.02; tunicamycin).
Main Limitations:
Mixed lamellar cell population, small sample size.
Conclusions:
ARAs reduced ER stress marker expression in ex vivo equine lamellar cells, suggesting potential for laminitis treatment and prevention.
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