β-Hydroxybutyrate in subclinical ketosis promotes endoplasmic reticulum biogenesis and milk protein synthesis via IRE1α-XBP1 in bovine mammary epithelial cells

Subclinical ketosis in dairy cows is accompanied by adaptive changes in the secretory function of the mammary gland. However, the molecular basis underlying this adaptation remains unclear. The secretory capacity of the mammary gland is closely linked to the activation of the unfolded protein response (UPR) and endoplasmic reticulum (ER) biogenesis, particularly the inositol-requiring enzyme 1α (IRE1α) pathway. Thus, this study aims to investigate whether BHB activates the IRE1α-XBP1 signaling axis to promote ER biogenesis, thereby sustaining casein synthesis. In this study, MAC-T cells were treated with 1.8 mM BHB for 0, 12, 24, or 48 h to mimic subclinical ketotic conditions. We first observed that BHB activated all 3 branches of the UPR without inducing the proapoptotic element of the UPR. Meanwhile, MAC-T cells treated with BHB show a significant increase in ER tracker staining and upregulated mRNA levels of ER biogenesis-related genes, such as choline kinase alpha (CHKA), choline-phosphate cytidylyltransferase alpha (PCYT1A), and Surfeit 4 (SURF4). Subsequently, BHB upregulated the mRNA abundance of genes related to ribosome biogenesis and the proregenerative phenotype in MAC-T cells. The MAC-T cells treated with 1.8 mM BHB also displayed increased protein abundance of β-casein, along with the casein beta (CSN2), casein kappa (CSN3), casein alpha S1 (CSN1S1), and casein alpha S2 (CSN1S2) milk protein genes. Compared with the 1.8 mM BHB group, cotreatment with BHB and KIRA6 significantly suppressed the BHB-induced increase in ER tracker fluorescence, ER biogenesis-related gene expression, and β-casein protein abundance. Silencing XBP1 via small interfering RNA inhibited BHB-induced ER biogenesis and β-casein synthesis, whereas the overexpression of XBP1 alleviated these effects. Furthermore, under BHB treatment, overexpression of XBP1 partially attenuated the suppressive effects of IRE1α inhibition on ER biogenesis and β-casein synthesis. Overall, our data demonstrate that BHB promotes ER biogenesis and milk protein synthesis in mammary epithelial cells through activation of the IRE1α-XBP1 signaling pathway, suggesting a protective and adaptive role for BHB in maintaining mammary gland function under subclinical ketotic conditions.