Mammary epithelial cells (MECs) have a distinct cell polarity, which is important for milk production during lactation. MECs take up glucose, amino acids, and fatty acids via transporters localized to their basolateral membranes [1,2], synthesize milk components in the endoplasmic reticulum (ER) or Golgi, and secrete them into the mammary alveolar lumen through the apical membranes via exocytosis or apocrine secretion [3,4]. This basal-to-apical directionality in MECs, which is essential during lactation, is established with the help of tight junctions (TJs).

TJs have three primary functions, namely, gate function, fence function, and epithelial polarity [5]. TJs in mammary glands during lactation comprise claudin-3 and occludin [6]. A subtype of claudin in the TJ region determines permeability through paracellular pathways [7]. TJs composed of claudin-3 exhibit less permeability and prevent the mixing of blood and milk, forming the blood–milk barrier [8]. By contrast, mammary glands afflicted by mastitis or going through involution express increased amounts of claudin-4, which weakens the TJ barrier function [9,10]. Apart from weakened TJs, the localization of aquaporin-3 transferred from the basolateral to the apical membrane in bovine MECs treated with lipopolysaccharides [2].

Epithelial cell functions are influenced by short-chain fatty acids (SCFAs). Stimulation with SCFAs enhances the TJ barrier function of intestinal epithelial cells [11]. SCFAs are the main energy sources for individual cells or tissues in ruminants such as cows and goats [12]. They regulate the production of milk components in MECs. Acetate or β-hydroxybutyrate (BHB), a butyrate metabolite, enhances the production of milk fat in bovine MECs [13]. Sodium butyrate (SB) also increases the content of antimicrobial components, such as β-defensin and S100A7, in milk produced by goat mammary epithelial cells (GMECs) [14].

However, some fatty acids induce ER stress in MECs [15], which can cause cellular dysfunction. The accumulation of unfolded or misfolded proteins causes ER stress and activates the unfolded protein response. Three signaling pathways have been characterized, namely, PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α, inositol-requiring protein 1α (IRE1α)–X-box-binding protein 1 (XBP1), and activating transcription factor 6α (ATF6α) [16,17]. Under ER stress, phosphorylated PERK activates ATF4 and ATF6α at the ER is trafficked into the nucleus. In addition, the activation of IRE1α cleaves a 26-nucleotide segment from unspliced XBP1 mRNA, introducing a translational frameshift that creates transcriptionally active XBP1 (S-XBP1), whose molecular weight is higher than that of unspliced XBP1 (U-XBP1, 54 kDa vs. 33 kDa, frame switch splicing) [18]. The activation of IRE1α also activates the nuclear factor-κB (NFκB) or c-Jun N-terminal kinase pathway [17].

Cells recognize and uptake SCFAs via G-protein-coupled receptors, such as GPR-41, -43, and -109A [[19], [20], [21]], and monocarboxylate transporters (MCTs) [22], which are localized to the apical and basolateral membranes in bovine MECs [23]. However, the influence of the directionality of SCFAs on the TJ barrier function in MECs remains unclear; thus, this study aimed to investigate the influence of sodium acetate and SB on the TJ barrier function in GMECs using an in vitro culture model, that exhibits distinct cell polarity [24].