PSVIII-1 Nutrient starvation induces autophagy and alters intestinal barrier function in IPEC-J2 cells

Abstract

Abstract Autophagy is a cellular process of controlled degradation of damaged organelles and cytoplasmic macromolecules during stress for maintaining homeostasis. Intestinal epithelial cells are barriers against microorganisms, toxins and food antigens. Whether autophagy plays a role in regulating intestinal barrier function is unclear. The objective of this study was to characterize the role of autophagy in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were nutrient starved in Krebs-Ringer bicarbonate (KRB) buffer for 0, 0.5, 1, 2, 3, 6, 9 and 12 h. Expression of genes implicated in autophagy regulation (AMPK, MDM2, p53 and DRAM) was determined by RT-PCR. The ratio of protein abundance of microtubule-associated protein light chain 3 (LC3-II/LC3-I) and p62, positive and negative markers of autophagy induction, respectively, was determined by western blotting. Compared with control group (0 h), the relative mRNA expression level of AMPK, MDM2, p53 and DRAM significantly decreased by an average of 52.5% (P < 0.01). Relative to 0 h, the mRNA expression of claudin 1 and claudin 4 significantly increased (108.0%) up to 6 h of starvation and then decreased (31.4%) thereafter (P < 0.01). On the contrary, abundance of claudin 1 and claudin 4 protein was downregulated (49.5%) up to 3 h of starvation and then increased (82.6%) thereafter (P < 0.01). Protein expression of claudin 3 was reduced (P < 0.01) with duration of starvation. There was no change in the protein level of occludin and ZO-1. The ratio of LC3-II/LC3-I significantly increased with duration starvation (P < 0.01), whereas p62 and phospho-AMPK levels decreased up to 6 h of starvation and then increased thereafter (P < 0.01). In summary, autophagy may be implicated in the regulation of tight junction integrity during nutrient starvation in IPEC-J2 cells.