Protective effects of adiponectin receptor agonists against equine lamellar endoplasmic reticulum stress

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] log 2 fold‐change [FC] = 4.8 [3.7–5.9]; p < 0.001; protein: log 2 FC = 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 log 2 FC = 3.1 [0.7–5.6]; p = 0.03) and interleukin‐8 (24 h; maximum log 2 FC = 5.4 [2.6–8.2]; p = 0.04) increased with both stressors. ER stress‐induced BIP protein expression was decreased by AICAR (log 2 FC = −2.0 [−5.1 to −1.1], p = 0.04) and AdipoRon (maximum log 2 FC = −1.8 [−2.1 to −1.5]; p = 0.001; tunicamycin), and by FLA (log 2 FC = −1.3 [−1.8 to −0.7]; p = 0.002), and gA (log 2 FC = −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 log 2 FC = −0.9; p < 0.001; tunicamycin). AdipoRon reduced RPS6 phosphorylation (log 2 FC = −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.