1. IntroductionType B trichothecenes constitute a class of mycotoxin frequently detected in cereals and grain products. Deoxynivalenol (DON) is the most commonly researched mycotoxin in the field of food safety. Its four structurally-related congeners 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), and nivalenol (NIV) are also regarded as important risk factors [1]. Many adverse effects caused by this family have been reported, such as anorexia, emesis, growth suppression, neuroendocrine changes, cytotoxicity, immunotoxicity, the inhibition of protein synthesis and mitochondrial translation [2,3,4,5]. DON and its congeners are well- known for causing decreased feed intake in human and a variety of animals [6,7,8,9] including broilers, pigs, dairy cow, mice and cats. The mechanism of type B trichothecene-induced anorexia is still not entirely clear.Food intake is influenced by both central factors and peripheral factors [10,11]. Anorexigenic molecules involving pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), melanocortin-3 receptor (MC3R), melanocortin-4 receptor (MC4R) and orexigenic molecule involving neuropeptide Y (NPY), and agouti-related peptide (AgPR) regulate appetite together. DON-induced anorexia is partly due to the up-regulation of central anorexigenic factors and the massive release of the peripheral satiety hormones, such as peptide YY(PYY) [12,13]. In addition, the release of some neurotransmitters located in the gastrointestinal (GI) tract play an important role in the regulation of anorexigenic signaling [14]. Our previous studies have shown that DON mediates anorexia by promoting the secretion of the neurotransmitters GLP-1 and GIP [15]. It has also been found that T2 toxins mediate anorexia by activating the secretion of the peptide neurotransmitter SP. Coincidentally, the secretion of both SP and GLP-1 can up-regulate the central anorexigenic factors [16]. However, the role of these two peptide neurotransmitters in type B trichothecenes-induced anorexia is not clear.SP is an undecapeptide of the neurokinin family, which abounds densely within nucleus tractus solitarii (NTS),the vagal and enteric nervous systems [17,18,19]. The food intake suppression of SP is involved in modulating the CRH signal and upregulating POMC expression [20,21,22]. GLP-1 is produced primarily by L cells in the distal ileum and colon, and is considered to action both as a peripheral satiety hormone and as a central neurotransmitter [23]. Studies of GLP-1 have expanded beyond glucose control to appetite regulation, energy balance and many more functions. The purpose of this study was to test the hypothesis that type B trichothecenes induce the release of SP and GLP-1 in mice and determine the role of these brain gut peptides in the anorectic effect of type B trichothecenes. Here, a proven murine anorexia model was used to relate SP and GLP-1 plasma concentrations using different methods (oral vs. IP) to five type B trichothecenes-induced food refusal. The SP and GLP-1 receptor antagonists were employed to verify the anorectic response. 2. ResultsAfter the oral and IP administration of DON, feed intake decreased significantly at 1 h (79% and 74%) and 2 h (75% and 69%), and showed a trend of recovery at 2–6 h after exposure, respectively (Figure 1A,D). Plasma SP concentration increased at 1 and 2 h, and returned to the initial concentration after 6 h (Figure 1B,E). Whereas, GLP-1 was only upregulated markedly at 2 h following IP exposure (Figure 1C,F).Food consumption was reduced significantly after the oral and IP administration of 3-ADON, e.g., DON at 1 h (72% and 75%) and 2 h (67% and 80%), and recovered at 6 h, respectively (Figure 2A,D). The concentrations of plasma SP (Figure 2B,E) and GLP-1 (Figure 2C,F) were significantly elevated by 3-ADON at 1 and 2 h, but returned to normal at 6 h.Additionally, 15-ADON induced anorexic responses similar to DON, with the feed intake significantly reduced at 1 h (76% and 54%) and 2 h (68% and 63%), and recovered at 6 h, with both oral and IP administration of 15-ADON (Figure 3A,D). Different from DON, plasma SP was elevated only at 2 h following oral exposure (Figure 3B,E). However, IP exposed to 15-ADON induced an increase in SP at 1 and 2 h. After 6 h, no difference in SP was observed. In contrast, plasma GLP-1 (Figure 3C,F) was significantly elevated by 3-ADON at 1 and 2 h after oral exposure. The concentration of plasma GLP-1 was robust upregulated only at 2 h through IP administration.FX caused rapid and prolonged (more than 6 h) anorexic responses following both oral and IP treatments (Figure 4A,D). Plasma SP (Figure 4B,E) was raised at 1 h and 2 h and still upregulated at 6 h. The concentration of plasma GLP-1 was increased at 2 and 6 h after oral exposure (Figure 4C,F), whereas IP exposed to FX evoked caused GLP-1 release at 1, 2 and 6 h. The prolonged elevations of SP and GLP-1 correlated with a persistent anorexic effect induced by FX.Unlike DON, NIV induced significant anorexia only at 2 h (56%) by oral administration (Figure 5A). Interestingly, plasma SP and GLP-1 concentrations increased significantly only at 2 h, consistent with the anorexic response (Figure 5B,C). After the IP administration of NIV, feed intake decreased significantly at 1 h (83%), 2 h (80%), and 6 h (59%) (Figure 5D). The concentration of plasma SP and GLP-1 also increased significantly at 1, 2 and 6 h (Figure 5E,F).Emend®, a receptor antagonist of NK-1R, was used to study SP- and DON-induced anorexia. Feed intake was significantly reduced at 1 h and 2 h after the IP administration of SP with 0.5 mg/kg BW and returned to normal at 6 h (Figure 6A). Serious anorexia occurred at 1 h, 2 h and 6 h after the IP administration of DON at 2.5 mg/kg BW (Figure 6B). Exposure to Emend® (1 mg/kg BW) alone had no effect on food consumption. Emend® attenuated SP- and DON-induced food refusal in a dose-dependent manner. Following the pretreatment of Emend® at 0.5 mg/kg BW, mice exposed to DON consumed 9, 26 and 37% more food at 1, 2 and 6 h, respectively. Mice receiving 1 mg/kg BW Emend® consumed 28, 59 and 45% more food at 1, 2 and 6 h than DON alone, respectively.Exending9-39, a receptor antagonist of GLP-1R, was used to study GLP-1- and DON- induced anorexia. Feed intake was significantly reduced at 1 h and 2 h after the IP administration of GLP-1 at 0.25 mg/kg BW and returned to normal at 6 h (Figure 7A). Serious anorexia occurred at 1 h, 2 h and 6 h after the IP administration of DON with 2.5 mg/kg BW (Figure 7B). Exposure to Exending9-39 (0.1 mg/kg BW) alone had no effect on food consumption. Exending9-39 attenuated SP- and DON- induced food refusal in a dose-dependent manner. Following the pretreatment of Exending9-39 at 0.05 mg/kg BW, mice exposed to DON consumed 52, 61 and 41% more food at 1, 2 and 6 h, respectively. Mice pretreated with 0.1 mg/kg bw Exending9-39 consumed 61, 64 and 50% more food at 0.5, 2 and 6 h than DON alone, respectively. 3. DiscussionMycotoxins in food and feed continue to threaten the health of humans and animals [24]. It is conservatively estimated that 25% of global food crops are contaminated with mycotoxins [25,26]. Type B trichothecenes are common in cereals. The mechanism behind the toxicity of DON, a type B trichothecene, has been extensively studied, but its four derivatives are often neglected. We integrated the data of trichothecene-induced anorexia of five type B trichothecenes in mice, and compared the changes in plasma concentrations of SP and GLP-1 with different administration methods (oral vs. IP). Additionally, DON-induced anorexia was inhibited by the SP receptor (NK-1 receptor) antagonist Emend® and the GLP-1 receptor antagonist Exendin9-39, confirming both brain-gut peptides may contribute to type B trichothecenes-induced food refusal in mice.Food consumption were markedly reduced with the IP administration of five type B trichothecenes. The same anorexia responses were observed after the oral administration of DON,3-ADON and 15-ADON; NIV was slightly different in that feed intake decreased significantly at 2 h, although there was also a downward trend in feed intake at 1 h compared with the control group after oral administration [27]. Consistent with previous research, five type B trichothecene-induced anorexia responses disappeared rapidly. The possible reason for this is that most neurotransmitters or satiety hormones which regulate appetite, are short-term [28,29].The plasma concentrations of SP with IP administration (five type B trichothecenes) and oral administration (DON,3-ADON, FX) were increased significantly at 1 h and 2 h. Moreover, 15-ADON and NIV, when orally administered, were a little different from the others; plasma SP increased significantly at 1 h and 2 h. SP is found in various tissues of the body [30] and the SP-induced anorectic response has been demonstrated [31]. Coincidentally, SP was first isolated from equine brain and gut [32]. Appetite and vomiting are regulated by SP through the central nervous system [33]. SP can also be secreted by gastrointestinal EC cells and bind to neurokinin 1 receptor (NK-1R) in the abdominal vagal nerve through paracrine, sending satiety signals via the brain-gut axis. The hypothalamus receives the signals and regulates appetite [34]. There is another interesting finding; SP is known as an antagonist of the GHSR-la receptor which inhibits ghrelin-induced food intake [35]. We hypothesize that five type B trichothecenes may promote gastrointestinal SP secretion to regulate feed intake.GLP-1 is released mostly by the L-cells of the intestines and is known for regulating energy balance (glucose homeostasis and appetite) via the peripheral and central systems [36,37]. The main function of GLP-1 is to inhibit the release of glucagon and stimulate the release of insulin [38]. An injection of GLP-1 into the lateral hypothalamus (LH), the core area for food intake, reduces food intake [39]. GLP-1 affects feed intake by regulating dopamine synthesis, the glutamate neurotransmitter and c-fos immunoreactivity [40,41,42]. The plasma concentrations of GLP-1 with oral administration and IP administration were increased significantly. Plasma GLP-1 with IP administration (DON, 15-ADON) and oral administration (FX, NIV) were increased significantly at 2 h. In addition, various bitter substances such as quinine, berberine and gentian root extract promote GLP-1 secretion by activating bitter taste receptor pathways [43,44,45]. Bitter substances are divided into five categories: terpenes and steroids, inorganic salts, alkaloids, flavanone glycosides, amino acids and peptides. These five type B trichothecenes belong to the terpene family. Therefore, we hypothesized that these five type B trichothecene-induced anorexia responses also promote GLP-1 release by activating the bitter taste receptor signaling pathway in the gut.Neurokinin receptors belong to the family of seven-transmembrane, G protein-coupled receptors, including NK1R, NK2R and NK3R, with SP preferring NK1R [46,47]. GLP-1 receptors are widely distributed in the hypothalamus, the vagal afferents, the area postrema (AP), the NTS and the intestine [48]. We found here that exogenous SP and GLP-1 induced food refusals, which could be inhibited by their antagonists, Emend® and Exendin9–39 [49,50]. NK1R and GLP-1R play an important role in DON-induced anorexia. 5. Materials and Methods 5.1. Animal and Reagent

B6C3F1 mice (10–11 weeks, female, Comparative Medicine Center of Yangzhou University) were individually housed in the room with a normal 12 h light/dark cycle. The room temperature and relative humidity were 20–23 °C and 40–60%, respectively. All guidelines for animal experiments followed the Institutional Animal Care and Use Committee at Nanjing Agricultural University. Type B trichothecenes were tested using a Liquid Chromatograph Mass Spectrometer and by conducting Elemental Analysis with a purity of more than 98%. SP (R&D Systems, Inc), GLP-1 and GLP-1R antagonist Exendin9-39 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in PBS. The NK-1R antagonist Emend® (Merck & Co, Inc) was dissolved in 1% DMSO in filter-sterilized PBS.

5.2. Experimental DesignThe design of the experiment with feed intake is shown in Figure 8A. The mice were randomly divided into groups based on body weight with 8 mice in each group and then acclimated to the environment for a week. The food of the mice was removed at 10:00 and the mice were fasted for 8 h on the day of the experiment. Then, 100 µL PBS or triclosporene B was gavaged orally or injected intraperitoneally with a dose of 2.5 mg/kg BW. The mice were given food pellets immediately and food intake was measured at 1, 2 and 6 h.The design of the study of brain–gut peptides is shown in Figure 8B. Groups of mice (n = 8/group) were orally or intraperitoneally administered 100 µL either PBS or type B trichothenenes at 2.5 mg/kg BW, respectively. Then, mice were anesthetized with sodium pentobarbital and sacrificed at 0, 1, 2 and 6 h. Plasma was collected through an EDTA anticoagulant tube and centrifuged for 10 min (3500× g, 4 °C). Brain–gut peptides SP and GLP-1 were determined by enzyme-linked immunosorbent assay (Phoenix Pharmaceutical).The experimental design for this study of brain–gut peptide receptor inhibitors is characterized in Figure 8C. Mice (n = 8/group) were orally given 100 μL of NK-1R antagonist Emend® or GLP-1R antagonist Exendin9-39 with 0, 0.5 and 1 mg/kg BW or 0, 0.05 and 0.1 mg/kg BW, respectively. After half an hour, mice were administrated an IP injection of SP or GLP-1 at 0.5 or 0.25 mg/kg BW in 100 μL, respectively. Control groups were first administrated either vehicle (1% DMSO) or antagonists orally (1 mg/kg BW Emend® or 0.1 mg/kg BW Exendin9-39), and then IP injected with PBS. Food intake was measured at 1, 2 and 6 h post-treatment.

To determine the role of SP or GLP-1 in DON and its congener-induced anorexia, mice were administrated 100 μL of antagonist orally (0, 0.5 and 1 mg/kg BW Emend® or 0, 0.05 and 0.1 mg/kg BW Exendin9-39) 30 min before oral exposure to 100 μL of DON at 2.5 mg/kg BW. Controls and food intake measurement time points were the same as the antagonist’s effect on SP and GLP-1-induced food refusal.

5.3. Statistics

All data were analyzed using SigmaPlot 11 for Windows (Jandel Scientific; San Rafael, CA, USA). Data are mean ± SEM (n = 8/group). Two-way repeated ANOVA (one factor) using the Holm–Sidak method was used to assess significant differences in food intake compared with the control at specific time points. Two-way ANOVA using Bonferroni t-test was used to analyze significant differences in the kinetics of SP and GLP-1 concentrations in plasma relative to the 0 h time point. One-way ANOVA using the Student–Newman–Keuls method was used to assess significant differences in food intake to determine the role of SP or GLP-1 in DON and its congener-induced anorexia. Significant differences were considered at p < 0.05.