Disruption of endoplasmic reticulum homeostasis exacerbates liver injury in clinically ketotic cows.

Disruption of endoplasmic reticulum (ER) homeostasis, a condition termed "ER stress," contributes to the development of liver injury in nonruminants. Because liver injury is a prominent pathological feature associated with overproduction of ketone bodies in dairy cows with ketosis, understanding the ER stress state and its functional consequences on liver injury is of particular interest. Here, 30 multiparous cows (within 3 wk postpartum) classified based on blood β-hydroxybutyrate (BHB) as healthy (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Compared with healthy cows, ketotic cows had greater levels of serum fatty acids and activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transferase, and glutamate dehydrogenase but lower serum glucose. Furthermore, dairy cows with ketosis had greater protein abundance of ER stress markers in liver tissue, including protein kinase RNA-like ER kinase (PERK), inositol-requiring protein-1α (IRE1α), and cleaved activating transcription factor-6 (ATF6). Cows with ketosis also had higher mRNA levels of hepatic 78-kDa glucose-regulated protein (GRP78) and spliced X-box binding protein 1 (sXBP1). These data confirmed an enhanced ER stress state in clinically ketotic cows. To explore whether enhanced hepatic ER stress was induced by elevated ketone bodies and the possible contribution of ER stress to liver injury, in vitro experiments were then performed using isolated primary calf hepatocytes treated with incremental concentrations of BHB (0, 0.6, 1.2, 3.0, and 4.8 mM) for 12 h with or without overexpression of GRP78 (the master regulator of unfolded protein response). Phosphorylation levels of PERK and IRE1α proteins, level of cleaved ATF6 protein, and mRNA abundance of GRP78 and sXBP1 in hepatocytes increased after treatment with high (3.0 and 4.8 mM) BHB, indicating a mechanistic link between excessive BHB and enhanced hepatic ER stress. Furthermore, treatment with 3.0 and 4.8 mM BHB markedly elevated activities of aspartate aminotransferase and alanine aminotransferase in cell supernatant, indicating exacerbated hepatocyte damage after ER stress was enhanced. Overexpression of GRP78 attenuated both BHB-induced ER stress and the ensuing cellular damage, suggesting that hepatocyte damage caused by excessive BHB can be mediated via enhanced ER stress. Overall, the present study revealed that ER stress may exacerbate liver injury development in clinically ketotic cows, underscoring the biological relevance of this pathway in the context of liver injury.