Leucine increases mucin 2 and occludin production in LS174T cells partially via PI3K-Akt-mTOR pathway.

Mucin 2 and occludin play a crucial role in preserving the intestinal mucosal integrity. However, the role for leucine mediating intestinal mucin 2 and occludin expression has little been investigated. The current study was conducted to test the hypothesis that leucine treatment could increase mucin 2 and occludin levels in LS174T cells. The LS174T cells were incubated in the Dulbecco's Modified Eagle Medium (DMEM) supplementing 0, 0.5 and 5 mmol/L L-leucine for the various durations. Two hours after the leucine treatment, the inhibitor of mammalian target of rapamycin (mTOR) and protein kinase B (Akt) phosphorylation in LS174T cells were significantly increased (P < 0.05), and the mucin 2 and occludin levels were also significantly enhanced (P < 0.05). However, the pretreatment of 10 nmol/L rapamycin, which was an mTOR inhibitor, or 1 μmol/L wortmanin, which was an inhibitor of phosphatidylinositol 3-kinase (PI3K), completely inhibited leucine-induced mTOR or Akt phosphorylation (P < 0.05), and significantly reduced leucine-stimulated mucin 2 and occludin levels (P < 0.05). These results suggest that leucine treatment promotes the mucin 2 and occludin levels in LS174T cells partially through the PI3K-Akt-mTOR signaling pathway.

Introduction

The mucus gel layer and the intercellular junctions between the intestinal epithelial cells are the crucial components of the non-specific barrier mechanisms in gut, which benefits the maintenance of intestinal mucosal integrity in human (Jankowski et al., 1994). The intercellular junctions between the intestinal epithelial cells mainly maintain through some transmembrane and non-membrane proteins, including occludin (Jankowski et al., 1994, Furuse et al., 1993). In addition, mucins, such as mucin 2, are secreted by goblet cells, which are the key components of the mucus gel layer in gut mucosa (Mao et al., 2011a). The intercellular junctions and mucins are important for maintaining animal and human health. Many studies have indicated that some nutrients, including essential amino acids, may affect the intestinal non-specific barrier mechanisms (Mao et al., 2011a, Mao et al., 2012, Chen et al., 2013, Wang et al., 2010). Our recent study also showed that dietary leucine supplementation significantly increased the mucin 2 level in the intestinal mucosa of weaned pigs (Mao et al., 2015). However, it is little known whether leucine can regulate the mucin 2 and occludin expression in intestinal cells.

The previous studies have also shown that leucine may stimulate specific protein expression in various cells (Zhang et al., 2014, Pérez de Obanos et al., 2006, Roh et al., 2003, Ijichi et al., 2003, Mao et al., 2011b). Moreover, during the protein synthesis in mammalian cells, leucine acts as the energy supply and substrates, and also regulates some intracellular signaling pathways, including the PI3K-Akt-mTOR signaling pathway (Kimball, 2002). LS174T cells can produce the tight junction protein (Elamin et al., 2014, Resta-Lenert et al., 2011), which is appropriate as a model to determine the barrier of function of the intestinal mucosa. Therefore, the present study hypothesized that leucine treatment can increase the mucin 2 and occludin levels in LS174T cells, and PI3K-Akt-mTOR pathway plays a potential role in regulating the mucin 2 and occludin levels by leucine.

Materials and methods

Cell culture

The LS174T cells from the American Type Culture collection (ATCC; Rockville, MD) were used as an in vitro model for intestinal epithelium. The cells (1 × 106 cells/well) were seeded in six-well plates in 2 mL of Dulbecco's Modified Eagle Medium (DMEM; Hyclone Laboratories Inc., Logan, UT) containing 4.5 g/L glucose, 4 mmol/L L-glutamine, 10% fetal calf serum (FCS; Gibco Laboratories Life Technologies Inc., Grand Island, NY) and antibiotics (100 units penicillin/mL and 100 μg streptomycin/mL; Gibco Laboratories Life Technologies Inc., Grand Island, NY) at 37°C and in a 5% CO2 atmosphere. When cells were grown to 90% confluence, they were starved for 12 h in serum and antibiotic-free DMEM.

Experiment one

The LS174T cells were incubated for 2 h in the presence of 0, 0.5 and 5 mmol/L leucine, and there were 6 replications for each leucine-treatment dose (n = 6). Following 2 h, the cells were collected and used to determine mTOR and Akt phosphorylation states and the levels of mucin 2, occludin and β-tubulin.

Experiment two

The time course (0, 1, 2, 4 and 6 h) of leucine treatment was administrated by incubating cells in the presence of the proper dose that was derived from Exp. 1, and there were 6 replications for each leucine-treatment duration (n = 6). Following the various culture durations, the cells were collected to examine mTOR and Akt phosphorylation states and the levels of mucin 2, occludin and β-tubulin.

Experiment three

After the starvation, LS174T cells were incubated in starvation media containing 10 nmol/L rapamycin or 1 μmol/L wortmanin. Following a 30 min treatment period, the proper dose of L-leucine that was derived from Exp. 1 was added to the media for the proper duration that was derived from Exp. 2, and there were 6 replications for each treatment (n = 6). Then, all cells were collected and used to determine mTOR and Akt phosphorylation states and the levels of mucin 2, occludin and β-tubulin.

The enzyme-linked immunosorbent assay (ELISA) analysis of mucin 2

The mucin 2 level was measured with ELISA analysis in the collected LS174T cells of each experiment, and there were 6 replications for each treatment (n = 6).

Cell protein isolation was performed as described previously (Mao et al., 2013). Briefly, following collection, the LS174T cells were suspended in 200 μL of fresh medium, and subjected to three cycles of freezing and thawing. The cell lysates were centrifuged at 10,000 × g and 4°C for 10 min. The supernatants were isolated and stored at −80°C until analyzed.

Then, mucin 2 protein levels were determined by ELISA as described previously with minor modifications (Devine et al., 1992, Elamin et al., 2014). Briefly, rabbit anti-mucin 2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was coated in the Falcon Microtest III ELISA 96-well plate (Becton–Dickinson, CA) by incubating 50 μL/well of a 1:100 dilution in the blocking solution (1% bovine serum albumin [BSA] in phosphate buffered saline [PBS]) overnight at 4°C. After expelling unbound antibody, the plate was blocked with 75 μL/well blocking solution at room temperature for 2 h. Following washing the plate 3 times with PBS containing 0.05% Tween-20, 50 μL/well of the cell protein sample diluted 1/2 in 2 × PBS containing 0.05% Tween-20 was added to each well and incubated overnight at 4°C. After washing the plate as above, mouse anti-mucin 2 (Abcam Inc., Cambridge, MA) was added by incubating 50 μL/well of a 1:100 dilution in the blocking solution at room temperature for 2 h. After washing the plate as above, goat anti-mouse horseradish peroxidase (HRP)-conjugated IgG (Jackson Immuno-Research Laboratories Inc., West Grove, PA) was added by incubating 25 μL/well of a 1:100 dilution in the blocking solution at room temperature for 1 h. Following washing the plate as above, 100 μL of 3,3′,5,5′-tetramethylbenzidine was added to each well and incubated with shaking in the dark at room temperature for 30 min. Finally, 25 μL of stop solution was added to each well, and the optical density was read at 450 nm using a BioTek Synergy HT microplate reader (BioTek Instruments, Winooski, VT).

Western blot analysis of occludin, β-tubulin and phosphorylation of mTOR and Akt

Protein levels for occludin, β-tubulin and the mTOR and Akt phosphorylation in LS174T cells were determined by Western blot analysis in the collected LS174T cells of each experiment, and there were 6 replications for each treatment (n = 6) as described previously with some modification (Mao et al., 2011b). Briefly, following lysing, the cell lysates were centrifuged for 15 min at 12,000 × g and 4°C. The supernatant was isolated. Protein concentration was determined using a BCA protein assay kit (Pierce Chemical Co., Rockland, IL). The supernatants containing equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 7.5% or 12% gel and transferred to polyvinylidene difluoride membranes. After the transfer, membranes were blocked with 5% non-fat dry milk in the buffer (0.1% Tween-20, 50 mmol/L Tris–HCl, pH 7.6, and 150 mmol/L NaCl) for 1 h at room temperature. Proteins were visualized with specific antibodies (Cell Signaling Technology, Beverly, MA), horseradish peroxidase conjugated secondary antibodies (Jackson Immuno-Research Laboratories Inc., West Grove, PA), and the Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The optical density of the bands for the occludin and the mTOR and Akt phosphorylation were normalized to their respective β-tubulin band using the Alpha Imager 2200 (Alpha Innotech, San Leandro, CA) software.

Statistical analysis

All data were expressed as means ± SE, analyzed using one-way ANOVA, and followed by Duncan's Multiple Range test. All analyses were performed using SAS (Version 8.1; SAS Institute, Cary, NC). P < 0.05 was considered statistical significance.

Results

Effect of leucine treatment on the phosphorylation of mTOR and Akt and the levels of mucin 2 and occludin

The levels of mTOR and Akt phosphorylation were significantly stimulated by the leucine treatment (P < 0.05), which changing was larger under 5 mmol/L leucine treatment (Fig. 1). Furthermore, as shown in Fig. 1, leucine treatment could significantly increase the levels of mucin 2 and occludin (P < 0.05), and that changing was also larger under 5 mmol/L leucine treatment (Fig. 1). Therefore, the dose of leucine treatment was 5 mmol/L in the remaining experiments of this study.

Fig. 1

Effect of supplementing different doses of leucine in medium on the phosphorylation of mammalian target of rapamycin (mTOR) and protein kinase B (Akt), and the levels of mucin 2 and occludin in LS174T cells. The LS174T cells were incubated with 0, 0.5 and 5 mmol/L leucine for 2 h. Representative western blots (A) for occludin, β-tubulin and phosphorylation of mTOR and Akt in LS174T cells were showed. Results were expressed as the amount of Akt phosphorylation (B), mTOR phosphorylation (C) and occludin (D) to β-tubulin and the amount of mucin 2 (E) in each treatment as a ratio of the treatment to the control. Values are means ± SE; n = 6. a,b,c Bars with different letters are significantly different (P < 0.05).

The mTOR and Akt phosphorylation levels were significantly stimulated at 1 h following 5 mmol/L leucine treatment (P < 0.05), and reached its maximum at 2 h (Fig. 2). Additionally, as shown in Fig. 2, the levels of mucin 2 and occludin were remarkably stimulated in 1 h following 5 mmol/L leucine treatment (P < 0.05), and reached a plateau by 2 h. Thus, the duration of leucine treatment was 2 h in the remaining experiments of this study.

Fig. 2

Effect of 5 mmol/L leucine treatment in medium on the phosphorylation of mammalian target of rapamycin (mTOR) and protein kinase B (Akt), and the levels of mucin 2 and occludin for the various duration in LS174T cells. The LS174T cells were incubated with 5 mmol/L leucine for 0, 1, 2, 4 and 6 h. Representative western blots (A) for occludin, β-tubulin and phosphorylation of mTOR and Akt in LS174T cells were showed. Results were expressed as the amount of Akt phosphorylation (B), mTOR phosphorylation (C) and occludin to β-tubulin (D) and the amount of mucin 2 (E) in each treatment as a ratio of the treatment to the control. Values are means ± SE; n = 6. a,b,c,d Bars with different letters are significantly different (P < 0.05).

Effect of leucine and/or inhibitor on phosphorylation of mTOR and/or Akt in LS174T cells

As shown in Fig. 3, the levels of mTOR phosphorylated on Ser2448 and Akt phosphorylated on Ser473 in LS174T cells were significantly increased by supplementing 5 mmol/L leucine (P < 0.05). Rapamycin, the inhibitor of the mTOR activation, significantly decreased the mTOR phosphorylation that was enhanced by supplementing leucine (P < 0.05, Fig. 3). Moreover, wortmanin, the phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly inhibited the Akt phosphorylation that was stimulated by supplementing leucine (P < 0.05, Fig. 3).

Fig. 3

Effect of rapamycin or wortmanin on phosphorylation of mammalian target of rapamycin (mTOR) or protein kinase B (Akt) in LS174T cells cultured in medium supplemented with leucine. The LS174T cells were subjected to pre-incubation with or without 10 nmol/L rapamycin (A) or 1 μmol/L wortmanin (B), and then treated with 5 mmol/L leucine for 2 h. Results are expressed as the amount of mTOR and Akt phosphorylation to β-tubulin in each treatment as a ratio of the treatment to the control. Values are means ± SE; n = 6. a,b,c Bars with different letters are significantly different (P < 0.05).

Effect of leucine and/or inhibitor on the mucin 2 level and the occludin expression in LS174T cells

As shown in Fig. 4, when LS174T cells were incubated in starvation media added 5 mmol/L leucine, the levels of mucin 2 and occludin were significantly enhanced (P < 0.05). However, the increasing level of mucin 2 and occludin in response to the addition of leucine was significantly inhibited by pretreatment with rapamycin or wortmanin (P < 0.05, Fig. 4).

Fig. 4

Effect of rapamycin or wortmanin on the mucin 2 and occludin levels in LS174T cells cultured in medium supplemented with leucine. The cells were subjected to pre-incubation with or without 10 nmol/L rapamycin (A and C) or 1 μmol/L wortmanin (B and D), and then treated with 5 mmol/L leucine for 2 h. Results are expressed as the amount of mucin 2 (A and B) and the amount of occludin to β-tubulin (C and D) in each treatment as a ratio of the treatment to the control. Values are means ± SE; n = 6. a,b,c,d Bars with different letters are significantly different (P < 0.05).

Discussion

Our previous study showed that dietary leucine supplementation could increase the number of goblet cell and the mucin 2 level in the jejunum of piglets (Mao et al., 2015). Similar to that study, the finding of the present work is that leucine treatment significantly increased mucin 2 and occludin levels in LS174T cells (Fig. 1, Fig. 2, Fig. 4). Furthermore, the effect of leucine treatment on mucin 2 and occludin levels might be relative to the PI3K-Akt-mTOR signaling pathway (Fig. 4).

Mucin 2, an important component of the mucus gel layer in the intestine, is mainly synthesized and secreted by the goblet cells, which is the important component of non-specific barrier mechanisms in the intestinal mucosa (Mao et al., 2011a). In addition, as a component of intestinal epithelial tight junction, occludin plays an important role in protecting animals and humans against enteric pathogens (Jankowski et al., 1994, Furuse et al., 1993). In the present study, leucine treatment significantly increased the mucin 2 and occludin levels in LS174T cells (Fig. 1, Fig. 2, Fig. 4). Recent studies have also shown that LS174T cells, a colorectal cell line derived from human, have the intestinal epithelial goblet-like characteristics, and can produce the tight junction protein, including occludin (Elamin et al., 2014, Resta-Lenert et al., 2011). Therefore, in the current study, we used LS174T cells as a model to determine the effect of leucine on mucin 2 and occludin levels in the intestinal mucosa. Therefore, it is possible that leucine supplementation might be able to improve the barrier function of intestinal mucosa via increasing the mucin 2 and occludin levels.

The previous studies have shown that leucine could increase the specific protein production in some cells via the PI3K-Akt-mTOR signaling pathway (Zhang et al., 2014, Pérez de Obanos et al., 2006, Roh et al., 2003, Ijichi et al., 2003, Mao et al., 2011b). In this study, the addition of leucine significantly increased the activity of PI3K-Akt-mTOR signaling pathway and the levels of mucin 2 and occludin (Fig. 1, Fig. 2, Fig. 4). In addition, rapamycin or wortmanin, the specific inhibitor of mTOR or PI3K, completely inhibited the phosphorylation of mTOR or Akt (Fig. 3), and significantly reduced the increase of mucin 2 and occludin levels that was promoted by leucine treatment (Fig. 4). Therefore, these results suggest that leucine increasing mucin 2 and occludin production of LS174T cells could at least partially be regulated by the PI3K-Akt-mTOR signaling pathway.

Conclusions

The results of the present study show that leucine treatment increased mucin 2 and occludin levels in LS174T cells through the PI3K-Akt-mTOR signaling pathway. However, the inhibitors of PI3K-Akt-mTOR signaling pathway could not completely inhibit the increase of mucin 2 and occludin levels that was promoted by leucine treatment, which reveals that leucine mediates the production of specific proteins possibly via other modes or pathways.

Conflict of interest

The authors declare that they have no conflict of interest.