In this study, we confirmed the hyperosmotic environment of intestinal lumen related to plasma as previously reported[16; 17], and it is a big challenge for intestinal epithelium. As for cells, hyperosmolarity shrinks cells by osmosis leading to the decrease of cell volume, the elevation of ROS and macromolecular crowding which are all candidates to be sensors triggering cell responses. With the underdeveloped structure, small intestine of piglets is more susceptible to hyperosmolarity so as to be damaged. In the present study, similar to other tissues or cells experiencing dramatic osmotic changes[26–28], betaine facilitates the adaption of porcine intestinal epithelial cells to environmental osmotic strength in vivo and vitro by counteracting the deleterious effect induced by hyperosmolarity.
Increased formation of ROS occurs when cells receive stress signaling. There were several researches reporting that hyperosmolarity caused oxidative stress with elevation of ROS[30–32], which was in agreement with our result. Meanwhile, we found that betaine decreased ROS accumulation in porcine intestinal epithelium in vivo and vitro, which was a good evidence for betaine to ameliorate hyperosmotic stress in porcine intestinal epithelium. Importantly, betaine functions as an osmoprotectant and helps to maintain cell volume, contributing to the decrease of hyperosmotic stress-induced ROS formation. Meanwhile, the methyl donor function of betaine may also contribute to the decrease of ROS. Betaine is capable of entering the SAAs (sulfur-containing amino acids) metabolism cycle via transmethylation with betaine-homocysteine methyltransferase (BHMT) (Supplemental Fig. 1) and consequently convert to SAAs including methionine, SAM and cysteine, which can relieve oxidative stress by scavenging ROS and increasing the production of GSH[33–37]. Sun et.al found that betaine prevented liver from oxidative injury by modulating the SAAs metabolism in which it increased the level of methionine, SAM and GSH. Moreover, the same result was also confirmed by Zhang et.al in vitro. In this study, it was worth noting that betaine supplementation significantly improved the expression of BHMT in intestinal epithelium of piglets (Supplemental Fig. 2) indicating the enhancement of transmethylation and SAAs metabolism in this trial. Thus, it is the promising next step to evaluate the antioxidative effect of betaine against hyperosmotic stress via SAAs metabolism with its three methyl groups.
Furthermore, we supported findings of others that hyperosmotic stress could lead to apoptosis[39–41], and betaine ameliorated hyperosmotic stress-induced mitochondrial dysfunction followed by apoptosis. Mitochondrial dysfunction is always an early event in hyperosmolarity-induced apoptosis, which can be caused directly by changes in matrix volume, secondary to the increased osmolality out of cell and hence in cytoplasm. Characterized with the permeabilization and depolarization of the outer membrane, mitochondrial membrane potential collapse is the first index of mitochondrial dysfunction and consequently results in the release of cytochrome c initiating caspases cascade reaction with the terminal of activated Caspase3. Bcl-2 belongs to Bcl-2 family and functions as a repressor to apoptosis by controlling the release of cytochrome c. Meanwhile, it is reported that Bcl-2 contributes to regulate excessive mitochondrial ROS-triggered promotion of apoptosis in a AMPK(AMP-activated protein kinase)-dependent way or by inducing the release of cytochrome c[44–46]. Consistent with researches in other tissue or cells[47; 48], our results demonstrated that hyperosmotic stress increased mitochondrial ROS and decreased mitochondrial membrane potential, which proved the hyperosmotic damage to mitochondria and the initiation of apoptosis. At the same time, the concentration of cytochrome c in cytoplasm and following cleaved Caspase3 rose accompanied with the decrease of the protein level of Bcl-2 under hyperosmotic condition, indicating the enhancement of mitochondrial-dependent apoptotic process in IPEC-J2 cells. However, betaine reversed the devastating effect of hyperosmolarity on mitochondria in this study so as to inhibit following apoptosis, at least, partly resulting from its osmoprotective role for maintaining mitochondrial homeostasis and its integrity.
Betaine alleviated the autophagy of porcine intestinal epithelium caused by hyperosmolarity. Upon hypertonic exposure, accumulation of ROS and molecular crowding together damage and aggregate proteins, which can activate cellular degradation mechanisms including autophagy. Autophagy, a type of programmed cell death, is a catabolic pathway involving self-degradation of damaged or aggregated protein or organelles and serves a critical role in cellular survival during pathophysiological processes[49; 50]. Since the autophagy is initiated, the cargo is sequestered into autophagosomes, the double-membrane vesicles tagged with lipid-conjugated microtubule-associated protein 1 light chain 3(LC3 II), and subsequently fuses with lysosomes forming autolysosomes to accomplish degradation. Sequestosome-1(SQSTM1/p62) is a ubiquitin-binding protein that promotes protein aggregate formation prior to their incorporation into autophagosomes. Thus, it serves as a key role in eliminating protein aggregate and it is negatively correlated with autophagy activation. Quite a few studies evaluating the role of hyperosmotic stress in autophagy induction in different species or tissues includes yeast, cardiomyocytes, rat notochordal cells, porcine renal proximal tubule-like(LLC-PK) cell. Here, we investigated the effect of hyperosmolarity on autophagy whose results were according with previous reports, as the increase expression of LC3 II and the decrease expression of p62 compared to the control in IPEC-J2 cells. Augment of autophagosomes and autolysosomes in hyperosmotic group was also a visible evidence of the enhancing autophagic flux. Milena Veskovic et al. found that betaine supplementation benefits MCD(methionine-choline deficient)-induced NAFLD (nonalcoholic fatty liver disease) mice by promoting autophagy. However, present studies showed that betaine alleviated hyperosmotic stress-induced autophagy with the decreased expression of LC3 II and increased expression of p62 in vivo and vitro. It can be on account of the compatible osmoregulatory properties of betaine that it not only protects cells from water loss but also stabilizes the structure of proteins under hyperosmotic condition. Instead of binding to the protein, betaine is excluded from a protein's hydration layer so that protein tends to fold up more compactly. Therefore, exposed to hyperosmolarity, betaine decreases the damaged proteins and inhibits the initiation of autophagy. Furthermore, we found that the decrease of autophagosomes was more dramatic than that of autolysosomes by betaine under hyperosmotic condition. It was suggested that betaine could reduce stimulus inducing autophagy such as protein damage and aggregate caused by hyperosmotic stress in cells without influencing the elimination of impaired content. Interestingly, although not achieving statistical significance, there was a decline of autophagy when cotreated with betaine in isosmotic medium in vitro as the lower level of LC3 II and higher level of p62. Based on the above result, we speculated that the effect of betaine on autophagy might be tissue-related, or other signaling pathway existed for inhibiting autophagy by betaine in addition to osmoprotective mechanism, which needed more exploration.