Docosahexaenoic and Eicosapentaenoic Acids Prevent Altered-Muc2 Secretion Induced by Palmitic Acid by Alleviating Endoplasmic Reticulum Stress in LS174T Goblet Cells

Diets high in saturated fatty acids (FA) represent a risk factor for the development of obesity and associated metabolic disorders, partly through their impact on the epithelial cell barrier integrity. We hypothesized that unsaturated FA could alleviate saturated FA-induced endoplasmic reticulum (ER) stress occurring in intestinal secretory goblet cells, and consequently the reduced synthesis and secretion of mucins that form the protective mucus barrier. To investigate this hypothesis, we treated well-differentiated human colonic LS174T goblet cells with palmitic acid (PAL)—the most commonly used inducer of lipotoxicity in in vitro systems—or n-9, n-6, or n-3 unsaturated fatty acids alone or in co-treatment with PAL, and measured the impact of such treatments on ER stress and Muc2 production. Our results showed that only eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids protect goblet cells against ER stress-mediated altered Muc2 secretion induced by PAL, whereas neither linolenic acid nor n-9 and n-6 FA are able to provide such protection. We conclude that EPA and DHA could represent potential therapeutic nutrients against the detrimental lipotoxicity of saturated fatty acids, associated with type 2 diabetes and obesity or inflammatory bowel disease. These in vitro data remain to be explored in vivo in a context of dietary obesity.


Introduction
Obesity is a complex and multifactorial pathology that is linked to low-grade systemic inflammation, which is identified as a key factor in its development and of related metabolic disorders [1,2]. Systemic inflammation has been shown to be closely related to intestinal microbiota dysbiosis during high-fat (HF) feeding in both mice [3] and humans [4]. A strong increase of gut permeability [5,6] associated with systemic inflammation is partly attributed to the downregulation of genes encoding tight junctions, such as zonula-occludens 1 and occludin [6,7]. A dense mucus layer protects the underlying colonic epithelium against injuries from luminal bacteria and the external environment [8]. The major macromolecular component of this thick barrier is the glycoprotein Muc2, produced by the intestinal goblet cells [9]. Muc2 contains a protein core with cysteine-rich and highly

Western Blot Analysis
LS174T cells were washed three times with ice-cold PBS and cell lysates were obtained in RIPA protein lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 1% NP (4-Nonylphenyl Poly(ethylene glycol)-40, 0.1% SDS, and 0.5% sodium deoxycholate, and a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, Saint Quentin Fallavier, France). The cells were kept for 15 min at 4 • C and disrupted by repeated aspiration through a 21-gauge needle. Cell lysates were sonicated and centrifuged at 15,000 g for 15 min at 4 • C and supernatants containing proteins were collected. The protein concentration was determined by a Bicinchoninic acid (BCA) assay. Thirty micrograms of total proteins were subjected to 12% polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to a Protan nitrocellulose membrane (Whatman, Dassel, Germany). After blocking non-specific binding sites with 5% free protease BSA in Tween-Tris-buffered saline (T-TBS, 0.1% Tween-20 in TBS) for 1 h, membranes were probed overnight at 4 • C under gentle agitation with primary CHOP and ATF4 antibodies (Cell Signaling, Ozyme, Saint-Cyr-l'École, France) and β-actin (C4) HRP (Santa Cruz Biotechnology, Heidelberg, Germany) at a concentration of 1/1000 and 1/5000, respectively. Blots were then washed three times in T-TBS for 10 min each, and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody at a concentration of 1/5000 for all the antibodies, except β-actin (C4), which was HRP conjugated. The detection of proteins was performed using the enhanced chemiluminescence (ECL) western blotting analysis procedure (Clarity Western ECL, Biorad, Marnes-la-Coquette, France), and their intensity was analyzed with the Chemidoc Imaging System (Biorad, Marnes-la-Coquette, France).

Muc2 ELISA Quantification
After the different treatments, the supernatant was centrifuged at 1000 g for 20 min at 4 • C to remove cells debris and 100 µL per sample was immediately used. The Muc2 Elisa Kit, purchased from Aviva Systems Biology (MUC2 ELISA Kit human, #OKEH02839, Clinisciences, Nanterre, France), was used to perform Muc2 quantification in the supernatant of LS174T cell cultures, following the manufacturer's instructions.

Statistical Analyses
One-way ANOVA was used to determine significance between different conditions. Tukey's multiple comparison test was used as a post-hoc comparison. For stimulation time-dependent gene expression data, one-way ANOVA with a Tukey's multiple comparison test as a post-hoc comparison was used to determine the significance difference of treatment at individual time points. The data collected for the two groups were analyzed by an unpaired two-tailed student T-test (* p < 0.05; ** p < 0.01; *** p < 0.001). GraphPad Prism version 7.00 (GraphPad Software Inc., San Diego, CA, USA) was used for the statistical analysis.

Effects of Palmitic Acid on Muc2 and KLF4 Expression and Muc2 Production in LS174T Cells
Since an HF diet (rich in PAL) was reported to induce a strong colon mucus layer thickness decrease in mice [11,39], we first wanted to assess the effects of PAL on Muc2 and KLF4 mRNA expressions and its impact on Muc2 secretion, the main mucin secreted by the LS174T cell line. We observed that 300 µM of PAL (considered a mildly elevated concentration) downregulated Muc2 mRNA expression after 6 and 24 h of treatment. Moreover, a significant decrease in goblet cell differentiation (KLF4) was observed after 3, 6, and 24 h of treatment with PAL ( Figure 1A). Furthermore, to determine whether PAL affects secretion of the Muc2 protein by LS174T cells, Muc2 quantification was performed in the culture medium after 24 h of treatment. The level of the mucous glycoprotein Muc2 was clearly reduced in the culture medium of LS174T cells treated with PAL for 24 h ( Figure 1B). These different data showed that PAL altered not only Muc2 and KLF4 expressions, but also Muc2 release by the LS174T cells, with no change in their viability at this concentration (300 µM) of PAL ( Figure S1). Furthermore, to determine whether PAL affects secretion of the Muc2 protein by LS174T cells, Muc2 quantification was performed in the culture medium after 24 hours of treatment. The level of the mucous glycoprotein Muc2 was clearly reduced in the culture medium of LS174T cells treated with PAL for 24 h ( Figure 1B). These different data showed that PAL altered not only Muc2 and KLF4 expressions, but also Muc2 release by the LS174T cells, with no change in their viability at this concentration (300 µM) of PAL ( Figure S1).

Effects of Palmitic Acid on Endoplasmic Reticulum Stress in LS14T Cells.
The high secretory output of the goblet cell makes Muc2, containing highly glycosylated domains, prone to misfolding, and failure to resolve this misfolding leads to ER stress, as shown in high-fat diets [11]. With this is mind, we investigated the impact of PAL on ER stress, by treating LS174T cells with PAL at various times ranging from 3 to 24 h and next evaluated markers of ER stress. ATF4 (activating transcription factor 4) and CHOP (CCAAT-enhancer-binding protein homologous protein) protein expressions were drastically increased in PAL-treated LS174T cells, whatever the duration of the treatment (Figure 2A). Comparable results were observed by the use

Effects of Palmitic Acid on Endoplasmic Reticulum Stress in LS14T Cells
The high secretory output of the goblet cell makes Muc2, containing highly glycosylated domains, prone to misfolding, and failure to resolve this misfolding leads to ER stress, as shown in high-fat diets [11]. With this is mind, we investigated the impact of PAL on ER stress, by treating LS174T cells with PAL at various times ranging from 3 to 24 h and next evaluated markers of ER stress. ATF4 (activating transcription factor 4) and CHOP (CCAAT-enhancer-binding protein homologous protein) protein expressions were drastically increased in PAL-treated LS174T cells, whatever the duration of the treatment (Figure 2A). Comparable results were observed by the use of thapsigargin as a positive control ( Figure 2B). Moreover, whatever the time of treatment, a clear increase in the mRNA expression of ER stress markers, GRP78 (glucose-related protein 78 kDa), and CHOP was observed in LS174T cells treated with PAL ( Figure 2C,D). ATF4 gene expression was increased at 6 h after PAL treatment, whereas it remained unchanged at 3 and 24 h ( Figure 2E).

Preventing Endoplasmic Reticulum Stress Restores Palmitic Acid-Altered Muc2 Secretion.
We next co-treated LS174T cells with PAL and 4-phenylbutyrate (4-PBA), an ER stress inhibitor, in order to verify whether altered Muc2 secretion is due to ER stress induced by PAL. After 24 h of treatment with PAL, the Muc2 concentration significantly decreased in the culture medium of LS174T cells, whereas co-treatment with 4-PBA normalized Muc2 secretion ( Figure 3). These results evidenced that the altered Muc2 release observed in PAL-treated LS174T cells mainly occurs through the triggering of ER stress.

Preventing Endoplasmic Reticulum Stress Restores Palmitic Acid-Altered Muc2 Secretion
We next co-treated LS174T cells with PAL and 4-phenylbutyrate (4-PBA), an ER stress inhibitor, in order to verify whether altered Muc2 secretion is due to ER stress induced by PAL. After 24 h of treatment with PAL, the Muc2 concentration significantly decreased in the culture medium of LS174T cells, whereas co-treatment with 4-PBA normalized Muc2 secretion ( Figure 3). These results evidenced that the altered Muc2 release observed in PAL-treated LS174T cells mainly occurs through the triggering of ER stress.

Only EPA and DHA Prevent Endoplasmic Reticulum Stress Induced by Palmitic Acid.
As n-3 fatty acids have been shown to prevent ER stress in both in vitro [36,40] and in vivo models [41][42][43], we investigated the interaction between PAL, n-3, n-6, and monounsaturated FA upon ER stress. As observed above (Figure 2), the protein expression levels of CHOP and ATF4

Only EPA and DHA Prevent Endoplasmic Reticulum Stress Induced by Palmitic Acid
As n-3 fatty acids have been shown to prevent ER stress in both in vitro [36,40] and in vivo models [41][42][43], we investigated the interaction between PAL, n-3, n-6, and monounsaturated FA upon ER stress. As observed above (Figure 2), the protein expression levels of CHOP and ATF4 were increased when LS174T cells were treated with PAL for 6 h. Moreover, co-treatment with PAL and -linolenic acid, linoleic acid, arachidonic acid, or oleic acid ( Figure 4A,B) also overexpressed ATF4 and CHOP protein expression. More interestingly, the upregulation observed with PAL was lowered only when PAL was combined with 25 µM of DHA or EPA ( Figure 4A). We next evaluated CHOP and ATF4 mRNA expressions in the same conditions. PAL overexpressed CHOP and similarly tended to increase ATF4, as already observed in Figure 2. Co-treatment of PAL with EPA and DHA alleviated CHOP and tended to reduce ATF4 overexpression, whereas LNA, OA, AA, and LA did not ( Figure 4C,D). Similar results were obtained after 3 and 24 h of treatment ( Figure S2).

EPA and DHA Prevent Palmitic Acid-Altered Muc2 and KLF4 Expressions and Muc2 Production in LS174T Cells.
As observed above ( Figure 1A), Muc2 and KLF4 mRNA expressions were downregulated when LS174T cells were treated with PAL for 6 h. Co-treatments with PAL and LNA, LA, AA, or OA also significantly decreased Muc2 and KLF4 mRNA expressions. Nevertheless, when EPA and DHA were added to PAL-treated LS174T cells, Muc2 and KLF4 mRNA expressions remained comparable to those of the control ( Figure 5A). Similar results were obtained at 24 h of treatment ( Figure S3). Lastly, when the level of the mucous glycoprotein Muc2 in the culture medium of LS174T cells was not affected by treatment with EPA, DHA, or AA alone, it was statistically

EPA and DHA Prevent Palmitic Acid-Altered Muc2 and KLF4 Expressions and Muc2 Production in LS174T Cells
As observed above ( Figure 1A), Muc2 and KLF4 mRNA expressions were downregulated when LS174T cells were treated with PAL for 6 h. Co-treatments with PAL and LNA, LA, AA, or OA also significantly decreased Muc2 and KLF4 mRNA expressions. Nevertheless, when EPA and DHA were added to PAL-treated LS174T cells, Muc2 and KLF4 mRNA expressions remained comparable to those of the control ( Figure 5A). Similar results were obtained at 24 h of treatment ( Figure S3). Lastly, when the level of the mucous glycoprotein Muc2 in the culture medium of LS174T cells was not affected by treatment with EPA, DHA, or AA alone, it was statistically reduced with PAL and with PAL and AA. In contrast, the secretion of Muc2 remained similar to the control when EPA and DHA were added to PAL ( Figure 5B).

Discussion
Obesity is now widely known to be associated with low-grade systemic inflammation. Indeed, it has been shown that HF diets, rich in saturated FA and particularly PAL, strongly increase intestinal permeability, leading to lipopolysaccharide absorption and metabolic endotoxemia that triggers inflammation and metabolic disorders [1,2,44]. The mucus layer, mainly comprised of the glycoprotein Muc2 produced by intestinal goblet cells [45], forming a physical barrier protecting the underlying epithelium against luminal substances and microbes [46,47], has been shown to be considerably altered under diets rich in saturated FA [11,18], exacerbating epithelium leakage and endotoxemia. We and others have evidenced that mice enriched in n-3 polyunsaturated fatty acids (PUFAs) are protected against gut barrier dysfunction, with consequences on metabolic endotoxemia [6,[48][49][50]. However, the effects of n-3 PUFAs on the secretory function of intestinal goblet cells remain largely unexplored. Using the well-differentiated human colonic goblet LS174T cell line, the present study shows that PAL decreases Muc2 production, mainly by generating a rise of ER stress in LS174T cells which detrimentally affects the production of the secreted mucosal barrier. More interestingly, we evidenced here, for the first time, that long-chain n-3 FA (C20 and C22 carbon chain length) are able to prevent the altered Muc2 production induced by PAL, mainly by alleviating ER stress.
Our results agree with previous reports showing that PAL induces ER stress in many cell types [33,34,51,52] and particularly with the one by Gulhane and co-workers, who showed that 500 µM of PAL induced significant stress in LS174T cells with a decrease in Muc2 and the goblet cell

Discussion
Obesity is now widely known to be associated with low-grade systemic inflammation. Indeed, it has been shown that HF diets, rich in saturated FA and particularly PAL, strongly increase intestinal permeability, leading to lipopolysaccharide absorption and metabolic endotoxemia that triggers inflammation and metabolic disorders [1,2,44]. The mucus layer, mainly comprised of the glycoprotein Muc2 produced by intestinal goblet cells [45], forming a physical barrier protecting the underlying epithelium against luminal substances and microbes [46,47], has been shown to be considerably altered under diets rich in saturated FA [11,18], exacerbating epithelium leakage and endotoxemia. We and others have evidenced that mice enriched in n-3 polyunsaturated fatty acids (PUFAs) are protected against gut barrier dysfunction, with consequences on metabolic endotoxemia [6,[48][49][50]. However, the effects of n-3 PUFAs on the secretory function of intestinal goblet cells remain largely unexplored. Using the well-differentiated human colonic goblet LS174T cell line, the present study shows that PAL decreases Muc2 production, mainly by generating a rise of ER stress in LS174T cells which detrimentally affects the production of the secreted mucosal barrier. More interestingly, we evidenced here, for the first time, that long-chain n-3 FA (C20 and C22 carbon chain length) are able to prevent the altered Muc2 production induced by PAL, mainly by alleviating ER stress.
Our results agree with previous reports showing that PAL induces ER stress in many cell types [33,34,51,52] and particularly with the one by Gulhane and co-workers, who showed that 500 µM of PAL induced significant stress in LS174T cells with a decrease in Muc2 and the goblet cell differentiation transcription factor KLF4 mRNA expressions [11]. These mRNA downregulations were also accompanied in the present study by a significant decrease of the secretion of the Muc2 protein by the cells (Figure 1). This result can be explained by the fact that PAL reduces the production of mature fully glycosylated Muc2 accompanied by an increase of the non-glycosylated Muc2 precursor and a reduction in mature Muc2 secretion, demonstrating protein misfolding consistent with the unfolded protein response (UPR) activation observed [11]. Due to their large size and complexity, mucins are extremely susceptible to misfolding in the ER, which can eventually lead to ER stress. We presently observed that PAL strongly increased ER stress in LS174T cells (Figure 2), which has also been observed with other ER stressors, such as thapsigargin, in the present study or tunicamycin in others [19]. In vivo, a decreased Muc2 secretion resulted in a thinner mucus layer more easily penetrated by diffusing microbial products, and more easily degraded by mucin-degrading bacteria. This is supported by previous studies evidencing that HF diet-induced obese mice exhibit a 50% thinner colon mucus layer [18]. Then, we may assume that the altered thickness of the mucus layer by an obesogenic diet (rich in PAL) could be explained by mucin misfolding leading to ER stress and an UPR-activated inhibition of Muc2 transcription with a downstream effect of reducing Muc2 production. This assumption is strengthened here by the use of the chemical ER chaperone 4-PBA (an ER stress inhibitor), which was able to prevent PAL-induced alteration of Muc2 production by LS174T cells (Figure 3).
To our knowledge, the present study is the first to demonstrate that ER stress produced by PAL in goblet LS174T cells can be significantly reduced by EPA and DHA, two highly unsaturated FA of the n-3 series exhibiting strong anti-inflammatory properties [53]. This is mechanistically supported by a decrease in the raised levels of UPR-related gene expression of CHOP and ATF4 associated with PAL in LS174T cells (Figure 4). In vitro studies have already suggested that DHA is able to counteract palmitate-induced ER stress in different cell types, including primary mouse hepatocytes [37], mouse 3T3L1 and rat primary preadipocytes [54], pancreatic cells [36,38], and C2C12 myotubes [40], but it has never been studied in colon cells in relation to mucus secretion. We evidenced in the present study that EPA and DHA are able to prevent altered Muc2 production in PAL-treated LS174T cells ( Figure 5B). This protective effect of EPA and DHA can be explained by ER stress alleviation, as shown Figure 4. The well-known anti-inflammatory effects of EPA and DHA can also be involved in the modulation of altered goblet cell homeostasis and the decreased production of mucin. Indeed, resolvins-derived from EPA and DHA-due to their anti-inflammatory properties, have also been suggested to attenuate ER stress-induced apoptosis in HepG2 cells, mainly through the JNK pathway [55]. Moreover, it has also been suggested that n-3 PUFA suppression of ER stress was partly due to AMP-activated protein kinase (AMPK) activation. In support of this, compound C (an AMPK inhibitor) is able to block the effects of DHA in PAL-induced ER stress inhibition [41].
LNA was supposed to have a similar efficiency to EPA and DHA against PAL lipotoxicity via reducing ER stress and apoptosis, as already shown in primary rat hepatocytes [56] or renal NRK-52E cells [57]. Unexpectedly, while LNA is also a n-3 fatty acid, it failed to prevent PAL alterations, as observed with EPA or DHA. Indeed, the expression of CHOP, ATF4, and Muc2 in LS174T cells co-treated with PAL and LNA remained similar to the one observed with PAL alone, whereas it was changed at least twice when cells were co-treated with PAL and EPA or DHA (Figures 4 and 5). Then, EPA, DHA, and LNA would be able to differently modulate PAL metabolism and consequently its lipotoxic effect. This has already been observed in other cell types, such as C2C12 myoblasts [58] and L6 myotubes [59], where partial and total oxidation were decreased during PAL treatment, which was restored by EPA and DHA, but not with LNA [58,59]. Then, we might expect comparable modulations in the present study. Moreover, besides alterations in its oxidation, it has also been shown that PAL treatment increased the formation of lipotoxic compounds, such as diglycerides and ceramides [60], and that EPA or DHA were able to 1) reduce such accumulation, 2) enhance triglyceride synthesis, and 3) preferentially address PAL to mitochondrial oxidation [58,61]. In contrast and in agreement with Pinel and coworkers [58], LNA failed to prevent PAL incorporation into cytotoxic diglycerides, and then to reduce the related ER stress activation and finally Muc2 production. Therefore, our results highly suggest that LNA intake in a diet high in n-3 fatty acids would not represent an alternative to oily fish consumption (rich in EPA and DHA) with regard to gut barrier integrity and especially to the preservation of the thickness of the mucus layer. In addition, a greater expression (+75%) of CHOP was observed in cells co-treated with PAL and AA compared with PAL alone (Figure 4). This increase could be due to the proinflammatory effects of AA-derived prostanoids and leukotrienes, as previously described [62], and supports the idea that certain n-6 fatty acids may have detrimental effects when consumed excessively.
In short, our results suggest that very long-chain n-3 PUFAs, by protecting goblet cells against ER stress-mediated altered Muc2 secretion induced by PAL, could be potential therapeutic nutrients used in strategies against the detrimental lipotoxicity of saturated FA, associated with type 2 diabetes and obesity or inflammatory bowel disease. Nevertheless, the relevance of our in vitro data remain to be explored in vivo in a context of dietary obesity, as well as the efficiency of n-3 PUFAs in alleviating the decrease of the thickness of the intestinal mucus layer and consequently preserving gut barrier integrity.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/9/2179/s1: Figure S1: Effect of palmitic acid and thapsigargin treatments on the viability of LS174T cells; Figure  Funding: This work was supported by the INSERM (Institut National de la Santé et de la Recherche Médicale), the Regional Council of Bourgogne, the European Regional Development Fund, the University of Bourgogne, the Fondation de France, and by a French Government grant managed by the French National Research Agency (ANR) under the program "Investissements d'Avenir" with the reference ANR-11-LABX-0021-01-LipSTIC LabEx. This work was also supported by Valorex (Combourtillé, France) and the Association Nationale Recherche Technologie (ANRT), who are financing Quentin Escoula with a CIFRE grant.