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High fat diet increases uptake of particles by the Peyer's patches of ileum in rats
Abstract
Background: Structure and functions of different organs especially cardiovascular, cerebrovascular and reproductive systems are compromised by the consumption of high fat diet. Gastrointestinal system has gained less attention in this respect. An intact and efficient gastrointestinal barrier protects us from different harmful agents. Objective: Our objective was to observe the effect of high fat diet on the uptake of particles by the peyer's patches (PP) and its transport to mesenteric lymph nodes of rats. Materials and Methods: Twenty, four weeks old male Wistar rats were used for the study. Rats were divided into two groups, control (n = 10) and HFD (n = 10) group. They received the respective diet for 6 weeks. 1 hour prior to sacrifice, 1 ml of fluorescent latex bead solution was given to all rats by gavage tube. After sacrifice, small intestinal samples containing PP were collected. Samples of ileum were fixed in formalin, cut in microtome for hematoxilin and eosin stain to observe and analyze small intestinal morphology. Ileum samples containing Peyer's patches and mesenteric lymph nodes were frozen in liquid nitrogen, cut in cryostat, taken on poly-l-lysine coated glass slides, observed and analyzed the number of fluorescent particles under confocal laser scan microscope. Statistical comparisons were performed by student's independent t test. Result: Body weight of the rats were significantly higher in HFD group as compared to the control group (C = 223 ± 25.6 gm, HFD = 339 ± 11 gm, p < 0.001). Height of the villi and crypt depth were significantly higher in HFD group as compared to the control group (C = 217 ± 7.7 μm, HFD = 324 ± 54.3 μm, p < 0.001 and 108 ± 7.6 μm, HFD = 136 ± 31.4 μm respectively). Uptake of particles by the PP was significantly higher in HFD group as compared to the control group (C = 23 ± 7.5, HFD = 106 ± 25.4, p < 0.001). Number of particles in the mesenteric lymph nodes was not different significantly between the two groups (C = 8 ± 3, HFD = 12 ± 3). Conclusion: We conclude that HFD for 6 weeks in rats increased the uptake of particles by the PP. We recommend that further studies should be conducted to find out the underlying mechanism to prevent or ameliorate such changes by several modulating agents. © 2013 Japan International Cultural Exchange Foundation & Japan Health Sciences University.
... In our previous study, we observed significantly higher uptake of fluorescent particles by the PP of rats fed HFD for 6 weeks [12]. But the underlying mechanism of this finding is unknown. ...
Prevalence of obesity is increasing worldwide. One of the major risk factors for obesity is consumption of high fat diet (HFD). Gastrointestinal tract (GIT) is the first organ where HFD comes in contact with the body. But, the effect of HFD on the GIT especially the GIT barrier is not investigated properly. M cells are present in the Follicle associated epithelium (FAE) of Peyer’s patches (PP) of GIT and are important component of intestinal barrier. Healthy and adequate number of M cells is important for an effective intestinal barrier. Intestinal tight junction protein Claudin 4, situated in between the enterocytes of Peyer’s patches (PP), regulates the permeability through the intestinal mucosa. Reduced Claudin 4 is responsible for increased paracellular transport of antigenic materials. Calprotectin is an inflammatory marker secreted by neutrophils during inflammation. Its level is considered specific for intestinal inflammation. The objectives of this study were to investigate the expressions of M cells and Claudin 4 in the PP and to determine the fecal calprotectin level (FCP) of male Wistar rats fed HFD. Four weeks old, twenty male Wistar rats were divided into chow (n=10) and HFD (n=10) groups. After 6 weeks of consuming the respective diets, stool and GIT segments containing PP were collected. After tissue processing, tissues were sectioned into 3 micrometer thickness and were taken on poly-L-lysine coated glass slides. Immunohistochemical staining was done by rat M cell specific CK-8 antibody and anti-Claudin 4 antibody. Scoring was done to calculate the average number of M cells and Claudin 4 in the PP of both groups under light microscope. FCP were measured using a commercial enzyme linked immunoassay (ELISA) kit. Statistical analysis was done by chi-square test and independent T-test. Data are presented as mean ± SD. A p value <0.05 was considered as significant. The number of M cells in the PP was significantly higher in HFD group as compared to the control (p = 0.004). The expression of Claudin 4 in the PP was significantly decreased in HFD group as compared to the control (p = 0.018). The fecal calprotectin level in HFD group was significantly higher compared to the control (p = 0.016). HFD consumption for 6 weeks leads to a higher number of M cell and reduced the expression of Claudin 4 in the intestinal Peyer’s patches of male Wistar rats which might be due to GI inflammation.
... Furthermore, the abnormal activation of resident intestinal immune cells by commensal bacteria might facilitate the development or persistence of intestinal inflammation. It was found that HFD caused a significant barrier decrease in rat PP [43] and induced translocation of pathogenic bacteria [10]. The HFD changes not only the PP bacteria from a taxonomic viewpoint, but also the phenotype of different immune modulating potentials. ...
Introduction: Zinc is essential for the development of enterocytes. Healthy enterocytes help to maintain efficient intestinal barrier to prevent excessive intestinal permeabilty to harmful agents. Chronic stress compromises morphology and permeability of the small intestine of rats. Objectives: To observe whether zinc supplementation can attenuate chronic stress induced small intestinal morphology and permeability changes in rats. Materials and methods: Forty male Sprague Dawley rats were divided into control (C), control with zinc CZ), stress (S) and stress with zinc (SZ) groups with ten rats in each group. Water avoidance stress was applied one hour daily for 10 consecutive days. Body weight gain, food and water intake and number of stool pellet after every stress session was measured. On the 11th day, fluorescent Isothiocyanate Dextran (FITC) was injected into the surgically created small intestinal loops. Blood was collect-ed one hour after the injection to measure serum FITC dextran. Jejunum and ileum was collected to measure morphological parameters. One way ANOVA was done to analyze data. Results: S group had significantly shorter crypt depth in ileum, shorter villus height in jejunum, fewer goblet cells in crypts of ileum and jejunum and fewer goblet cells in villus of jejunum as compared to the C group. SZ group had significantly longer crypt depth, longer villus height and higher number of goblet cells in crypts and villus of ileum and jejunum as compared to the S group. S group had significantly higher permeability to FITC and increased inflammatory cell infiltration in ileum and jejunum as compared to the C group. SZ group had significantly lower permeability to FITC and fewer inflammatory cells infil-tration in ileum and jejunum as compared to the S group. Conclusion: Chronic stress compromised the small intestinal morphology and permeability which were attenuated by zinc supplementation.
Obesity is a predictor of diabetes and cardiovascular disease. One consequence of obesity is dyslipidemia characterized by high blood triglycerides. It has been proposed that oxidative stress, driven by utilization of lipids for energy, contributes to these diseases. The effects of oxidative stress are mitigated by an endogenous antioxidant enzyme network, but little is known about its response to high fat utilization. Our experiments used a multiplexed quantitative proteomics method to measure antioxidant enzyme expression in heart tissue in a mouse model of diet-induced obesity. This experiment showed a rapid and specific up-regulation of catalase protein, with subsequent assays showing increases in activity and mRNA. Catalase, traditionally considered a peroxisomal protein, was found to be present in cardiac mitochondria and significantly increased in content and activity during high fat feeding. These data, coupled with the fact that fatty acid oxidation enhances mitochondrial H2O2 production, suggest that a localized catalase increase is needed to consume excessive mitochondrial H2O2 produced by increased fat metabolism. To determine whether the catalase-specific response is a common feature of physiological conditions that increase blood triglycerides and fatty acid oxidation, we measured changes in antioxidant expression in fasted versus fed mice. Indeed, a similar specific catalase increase was observed in mice fasted for 24 h. Our findings suggest a fundamental metabolic process in which catalase expression is regulated to prevent damage while preserving an H2O2-mediated sensing of diet composition that appropriately adjusts insulin sensitivity in the short term as needed to prioritize lipid metabolism for complete utilization.
Background: High dietary fat is linked to cardiac oxidative stress, however, little is known about the endogenous antioxidant response.
Results: High fat feeding and fasting rapidly up-regulate catalase.
Conclusion: Up-regulation of catalase is designed to protect mitochondria from oxidative damage while not perturbing H2O2-mediated signaling.
Significance: Coupling fatty acid oxidation to H2O2 production creates a mechanism for sensing and communicating diet composition.
It has been demonstrated that the type of diet affects the brain structure and function. Consumption of fat-rich food is one of the most important factors that lead to increase in the prevalence of cardiovascular and neurological diseases. High-fat diet may change the volume and neuronal number or density in the hypothalamus, which is the center of energy control. Therefore, this study was designed to study the effect of high-fat diet on the density and number of neurons, and also the volume of hypothalamus in adult male mice. Forty male mice were divided into the control and experimental groups. The control group were fed with standard and the experimental groups, with high-fat diet for 4 (short-term) or 8 (long-term) weeks. The animals were perfused and brains were immediately removed, post-fixed and cut coronally and serially using cryostat at 30-µm thickness. Every 6th sections were stained by cresyl violet. The numerical density and number of neuron and the volume of hypothalamus were estimated by using unbiased stereological methods. Data analysis showed that both short and long time consumption of high-fat diet decreased the neuronal cell density of the hypothalamus. Interestingly, despite a decrease in the neuronal cell density, long time consumption of high-fat diet could significantly increase the volume of hypothalamus (P<0.05). High fat diet decreased the neuronal cell density and increased the volume of the hypothalamus, but it did not significantly change its total neurons. These changes might be due to an increase in the extracellular space through inflammation or gliosis in the hypothalamus.
Gastrointestinal (GI) motility and gut hormones have been considered to be involved in the development and maintenance of obesity. Our aim was to assess the relationships between gastric emptying (GE), GI transit and gut hormones and leptin concentrations in diet-induced obese rat model. Male 6-week-old Sprague-Dawley rats were fed with a high-fat (HF) diet for 8weeks to generate diet-induced obesity (DIO) and diet resistant (DR) rats. GE, GI transit and plasma ghrelin, cholecystokinin (CCK), PYY and leptin concentrations were determined in DIO, DR and control (CON) rats. The DIO rats had slower GE, higher plasma leptin and CCK concentrations, and lower plasma ghrelin concentration compared with CON and DR rats. GE was correlated with plasma ghrelin (r=0.402, P=0.028), CCK (r=-0.518, P=0.003) and leptin concentration (r=-0.514, P=0.004). The slower GE, which can be considered as an adaptive response aimed at HF diet induced obesity, may be mediated by changes of plasma ghrelin, CCK and leptin concentrations.
Sprague-Dawley rats (3 weeks old) were fed on isoenergetic diets in which 40% of the total energy was provided as fat either in the form of butter (high saturated fat), olive oil (high monounsaturated fat) or maize oil (high polyunsaturated fat), with one group on low-fat (10% of total energy) standard diet as a control. Animals were killed after 8.4 (se 0.8) weeks by cardiac puncture. Similar pieces of jejunum and ileum were prepared for morphometric studies. Extracts of tissue from the proximal and distal segments of the whole small intestine from four animals per group were assayed using established techniques for enteroglucagon, motilin, neurotensin, somatostatin, substance P and vasoactive intestinal peptide (VIP). We found that maize oil and olive oil increased villus height: crypt depth ratio in both jejunum and ileum. Maize oil increased tissue concentrations of somatostatin (P less than 0.05) and substance P (P less than 0.005) in the proximal segment. Both maize oil and olive oil increased tissue concentrations of neurotensin and substance P (P less than 0.005) in the distal segments. These observations may explain the improvement of intestinal absorption of fluid following supplementation with polyunsaturated fat.
Membranous epithelial or "M" cells in lymphoid follicle epithelium of Peyer's patches extend between the adjacent columnar cells, forming a membrane separating lymphocytes in the epithelial cell layer from the intestinal lumen. They lack developed microvilli, glycocalyx, or terminal web but contain numerous vesicles. Transport of particles from ligated intestinal loops by such cells without evidence of further uptake of these particles by lymphocytes has been described by others. Horseradish peroxidase (HRP), an enzyme known to function orally and parenterally as a foreign protein antigen, was injected into the unligated intestines of fasted healthy 2-month-old white Swiss mice to determine if HRP would be absorbed in detectable quantities by M cells, and whether subsequent uptake of HRP by lymphocytes could be documented. At intervals from 1 to 60 min the most distal ileal Peyer's patch was fixed, removed, reacted with H2O2-3,3'-diaminobenzidine and examined by light and electron microscopy for HRP reaction product. At 1 min HRP adhered to surfaces of columnar cells and M cells and extended down into surface pits in the M cell. After 5 min HRP was found in vesicles of M cells but not in columnar cells. At 1 hr HRP was detected in the extracellular space between M cells and their enfolded lymphocytes, as well as in vesicles within these lymphocytes. Transport of intestinal luminal material by M cells with subsequent uptake by lymphocytes provides a specific route for antigen uptake into the intestinal lymphoid system.
Uptake and translocation of particulates across the mucosal barrier of the gastrointestinal (GI) tract is now generally recognised but the effect of pathophysiologically induced changes on this process is less well established. This study evaluated the effect of diabetes mellitus on GI absorption of particles, comparing particle localisation and particle loading in different microanatomical sites of the primary organ (small intestine) and possible particle translocation pathways to selected secondary organs (mesenteric lymph nodes, liver, spleen) in normal and streptozotocin-induced diabetic animals. Fluorescent polystyrene latex particles (approximately 2 microns diameter) were fed orally to young adult Sprague-Dawley rats and quantitative bulk tissue and morphological techniques used to chart particle transit across the small intestine to secondary organs 0.5 h postadministration. In the normal animal, epifluorescence and confocal laser scanning microscopy provided confirmatory evidence for particle absorption within the primary organ and transport to other sites in the body. By contrast, in the diabetic animal, particle translocation and peripheral distribution were reduced with approximately 30% decrease in particle loading in the epithelial/nonepithelial tissue compartments. This could be a consequence of gastric retention and altered intestinal motility and permeability which are known to be associated with diabetes.
Dysfunction of the intestinal barrier, as evidenced by increased intestinal permeability and bacterial translocation, has been reported under total parenteral nutrition (TPN). However, the role of Peyer's patches on the intestinal barrier in TPN is not well understood. We investigated whether TPN alters the uptake of microparticles by the follicle-associated epithelium of Peyer's patches.
Twenty rats were divided into two groups, a control group and a TPN group. Fluorescent polystyrene latex beads, 3.2 +/- 0.2 microns in diameter, were used as a probe for measuring the uptake by Peyer's patches. After 1 week of consuming either the control or TPN diet, rats were killed. On the day of killing, 0.1 mL of latex beads solution was injected into a 1-cm length of ileal loop, within 10 cm of the ileocecal valve. Samples were taken after 30 minutes of injection, sectioned by cryostat, and then viewed under a fluorescent microscope. Follicle-associated epithelial length and particles were counted using a confocal laser scanning microscope. The number of particles within each compartment was standardized per unit length of epithelium of Peyer's patches.
Particle numbers within Peyer's patch dome of the TPN group were significantly increased compared with those of the control group (p < .01).
These data suggest that dysfunction of the intestinal barrier in TPN might be associated with a change of uptake by Peyer's patches.
Effect of supplemental alanyl-glutamine in standard TPN (S-TPN) on luminal mucus gel and small intestinal permeability was investigated.
Thirty Sprague-Dawley rats were divided into group I (n = 10), receiving standard rat diet; group II (n = 10), receiving S-TPN; and group III (n = 10), receiving alanyl-glutamine-supplemented TPN for 1 week. After 1 week, fluorescein isothiocyanate (FITC)-dextran was injected into the small intestine of the rats, and they were killed. A small intestinal sample and portal blood were obtained for morphologic and functional analysis of mucus gel and intestinal permeability.
In group II, thickness and optical density of mucus gel per millimeter serosal length of intestine were significantly lower than group I (p<.001) and were significantly higher in group III than in group II (p<.001). The number of goblet cells in the villi and in the crypt of the small intestine was significantly lower in group II than in group I (p<.001) and was significantly higher in group III than in group II (p<.001), with the exception of the villi of jejunum. Villous and crypt surface area per millimeter serosal length of intestine was significantly lower in group II than in group I (p<.001) and was significantly higher in group III than in group II (p<.001). Small intestinal permeability to FITC-dextran was significantly higher in group II than in group I (p<.001) and was significantly lower in group III than in group II (p<.001). Glucosamine synthetase level was significantly higher in group III than in group I and ileum of group II (p<.001).
Alanyl-glutamine-supplemented TPN prevents a decrease in mucus gel and an increase in small intestinal permeability associated with S-TPN.
M cells, an epithelial cell phenotype that occurs only over organized mucosal lymphoid follicles, deliver samples of foreign material by transepithelial transport from the lumen to organized lymphoid tissues within the mucosa of the small and large intestines. The apical membranes of M cells in the intestine are designed to facilitate adherence and uptake of antigens and microorganisms, a prerequisite for immunological sampling. The molecular features of M cell apical surfaces that promote adherence and transport are crucial for understanding the strategies that pathogens use to exploit this pathway.
M cells play an important role in the intestinal immune system as they have a high capacity for transcytosis of a wide range of microorganisms and macromolecules. However, little is known about the role of M cells during intestinal inflammation.
We studied M cell development during indomethacin-induced intestinal inflammation in rats.
Ileitis in rats was induced by two subcutaneous injections with indomethacin (7.5 mg/kg) given 24 h apart. Rats were sacrificed after 14 days and tissue was analysed by fluorescence microscopy and electron microscopy. M cells could be visualized by using the FITC-labelled mAb anti-cytokeratin (CK)-8 (clone 4.1.18), which was recently identified as specific M cell marker in rats. The number of cytokeratin-8 positive M cells was related to the surface of the follicle associated epithelium. For morphological studies, we used both transmission electron microscopy (T.E.M.) and scanning electron microscopy (S.E.M.).
In non-inflamed ileum M cells were scarce. Only 4% of the follicle associated epithelium were M cells, whereas an increase of M cells up to 11% was found in inflamed follicle associated epithelium (P < 0.001). The rate of M cell induction depended on the macroscopic degree of inflammation. T.E.M./S.E.M. studies showed that in inflamed tissue most M cells underwent apoptosis with typical morphological signs. In contrast to apoptotic M cells, the neighbouring enterocytes usually appeared intact. The number of mononuclear cells below the follicle associated epithelium was significantly increased. S.E.M. studies revealed that during induced ileitis mononuclear cells migrated from the lamina propria into the gut lumen by passing through apoptotic M cells.
During indomethacin-induced ileitis in rats the increase in M cell number in association with apoptosis of M cells may alter the intestinal barrier function. These observations may play a pivotal role in the pathogenesis of chronic intestinal inflammation, e.g. in inflammatory bowel disease.
Gut-associated lymphoreticular tissues, such as Peyer's patches and cecal patches, are important inductive sites for mucosal immune responses. As such, gut-associated lymphoreticular tissues may have an epithelial barrier different from that of villous epithelium. In this study, we investigated the immunohistochemical distribution of the claudin family and occludin in the follicle-associated epithelium (FAE) of Peyer's patches and cecal patches of murine intestine. Unique profiles of claudin-2, -3, and -4 and occludin expression were noted in the tight junctions of the FAE: claudin-4 was preferentially expressed in the apex region; claudin-2 was only weakly expressed on the crypt side of the FAE compared with stronger expression on the crypt side of villous epithelial cells; and claudin-3 and occludin were found throughout the dome. These unique expression patterns were present also in cecal patch FAE. We also found that claudin-4 expression in the FAE of Peyer's patches and cecal patches correlated with the presence of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)-positive apoptotic cells, and Peyer's patch-deficient mice exhibited expression patterns of claudin and occludin in villous epithelia similar to those in wild-type mice. We conclude that claudin-4 expression was preferentially associated with the dome region of FAE, the mucosal inductive site of the murine intestine. In that location it might correlate with the cell life cycle, help maintain the apex configuration of the dome, or be a factor favoring the uptake of antigens by the FAE.
Chronic stress affects the course of inflammatory bowel disease and experimental colitis, and may also initiate intestinal inflammation in rats.
To investigate the effects of stress on the M cell containing follicle associated epithelium, specialised in antigen uptake.
Wistar rats were submitted to acute water avoidance stress for one hour or chronic water avoidance stress for 1 hour/day for 10 consecutive days. Permeability to (51)Cr-EDTA, horseradish peroxidase, and chemically killed Escherichia coli K-12 was studied in both villus and follicle associated epithelium in Ussing chambers. Segments were further examined by light, electron, and confocal microscopy.
Acute stress increased horseradish peroxidase flux in villus as well as in follicle associated epithelium. Chronic stress further increased permeability to horseradish peroxidase in villus and follicle associated epithelium, in the latter by almost fourfold. Moreover, chronic stress induced over 30 times increased E coli passage in follicle associated epithelium whereas there was no significant increase in villus epithelium. Bacterial uptake was confirmed by confocal microscopy showing fluorescent bacteria penetrating and passing through the epithelial surface.
These results show that the barrier function of follicle associated epithelium can be modulated, and that chronic stress enhances the uptake of luminal antigens and bacteria via the follicle associated epithelium. This can increase antigen exposure in Peyer's patches thereby having implications in the initiation of proinflammatory immune responses within the intestinal mucosa.