Anatomy and Neuroscience - Research Publications

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    Selenium and vitamin E together improve intestinal epithelial barrier function and alleviate oxidative stress in heat-stressed pigs
    Liu, F ; Cottrell, JJ ; Furness, JB ; Rivera, LR ; Kelly, FW ; Wijesiriwardana, U ; Pustovit, RV ; Fothergill, LJ ; Bravo, DM ; Celi, P ; Leury, BJ ; Gabler, NK ; Dunshea, FR (WILEY, 2016-07-01)
    What is the central question of this study? Oxidative stress may play a role in compromising intestinal epithelial barrier integrity in pigs subjected to heat stress, but it is unknown whether an increase of dietary antioxidants (selenium and vitamin E) could alleviate gut leakiness in heat-stressed pigs. What is the main finding and its importance? Levels of dietary selenium (1.0 p.p.m.) and vitamin E (200 IU kg(-1) ) greater than those usually recommended for pigs reduced intestinal leakiness caused by heat stress. This finding suggests that oxidative stress plays a role in compromising intestinal epithelial barrier integrity in heat-stressed pigs and also provides a nutritional strategy for mitigating these effects. Heat stress compromises the intestinal epithelial barrier integrity of mammals through mechanisms that may include oxidative stress. Our objective was to test whether dietary supplementation with antioxidants, selenium (Se) and vitamin E (VE), protects intestinal epithelial barrier integrity in heat-stressed pigs. Female growing pigs (n = 48) were randomly assigned to four diets containing from 0.2 p.p.m. Se and 17 IU kg(-1) VE (control, National Research Council recommended) to 1.0 p.p.m. Se and 200 IU kg(-1) VE for 14 days. Six pigs from each dietary treatment were then exposed to either thermoneutral (20°C) or heat-stress conditions (35°C 09.00-17.00 h and 28°C overnight) for 2 days. Transepithelial electrical resistance and fluorescein isothiocyanate-dextran (4 kDa; FD4) permeability were measured in isolated jejunum and ileum using Ussing chambers. Rectal temperature, respiratory rate and intestinal HSP70 mRNA abundance increased (all P < 0.001), and respiratory alkalosis occurred, suggesting that pigs were heat stressed. Heat stress also increased FD4 permeability and decreased transepithelial electrical resistance (both P < 0.01). These changes were associated with changes indicative of oxidative stress, a decreased glutathione peroxidase (GPX) activity and an increased glutathione disulfide (GSSG)-to-glutathione (GSH) ratio (both P < 0.05). With increasing dosage of Se and VE, GPX-2 mRNA (P = 0.003) and GPX activity (P = 0.049) increased linearly, the GSSG:GSH ratio decreased linearly (P = 0.037), and the impacts of heat stress on intestinal barrier function were reduced (P < 0.05 for both transepithelial electrical resistance and FD4 permeability). In conclusion, in pigs an increase of dietary Se and VE mitigated the impacts of heat stress on intestinal barrier integrity, associated with a reduction in oxidative stress.
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    Glucagon-like peptide 1 and peptide YY are in separate storage organelles in enteroendocrine cells
    Cho, H-J ; Robinson, ES ; Rivera, LR ; McMillan, PJ ; Testro, A ; Nikfarjam, M ; Bravo, DM ; Furness, JB (SPRINGER, 2014-07)
    A sub-group of enteroendocrine cells (L cells) release gastrointestinal hormones, GLP-1 and PYY, which have different but overlapping physiological effects, in response to intraluminal nutrients. Whilst their release profiles are not identical, how the plasma levels of these two hormones are differentially regulated is not well understood. We investigate the possibility that GLP-1 and PYY are in separate storage vesicles. In this study, the subcellular location of GLP-1 and PYY storage organelles is investigated using double-labelling immunohistochemistry, super resolution microscopy and high-resolution confocal microscopy. In all species tested, human, pig, rat and mouse, most cytoplasmic stores that exhibited GLP-1 or PYY immunofluorescence were distinct from each other. The volume occupancy, determined by 3D analysis, overlapped by only about 10∼20 %. At the lower resolution achieved by conventional confocal microscopy, there was also evidence of GLP-1 and PYY being in separate storage compartments but, in subcellular regions where there were many storage vesicles, separate storage could not be resolved. The results indicate that different storage vesicles in L cells contain predominantly GLP-1 or predominantly PYY. Whether GLP-1 and PYY storage vesicles are selectively mobilised and their products are selectively released needs to be determined.
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    Effects of Food Components That Activate TRPA1 Receptors on Mucosal Ion Transport in the Mouse Intestine
    Fothergill, LJ ; Callaghan, B ; Rivera, LR ; Lieu, T ; Poole, DP ; Cho, H-J ; Bravo, DM ; Furness, JB (MDPI AG, 2016-10)
    TRPA1 is a ligand-activated cation channel found in the intestine and other tissues. Components of food that stimulate TRPA1 receptors (phytonutrients) include allyl isothiocyanate, cinnamaldehyde and linalool, but these may also act at other receptors. Cells lining the intestinal mucosa are immunoreactive for TRPA1 and Trpa1 mRNA occurs in mucosal extracts, suggesting that the TRPA1 receptor is the target for these agonists. However, in situ hybridisation reveals Trpa1 expression in 5-HT containing enteroendocrine cells, not enterocytes. TRPA1 agonists evoke mucosal secretion, which may be indirect (through release of 5-HT) or direct by activation of enterocytes. We investigated effects of the phytonutrients on transmucosal ion currents in mouse duodenum and colon, and the specificity of the phytonutrients in cells transfected with Trpa1, and in Trpa1-deficient mice. The phytonutrients increased currents in the duodenum with the relative potencies: allyl isothiocyanate (AITC) > cinnamaldehyde > linalool (0.1 to 300 μM). The rank order was similar in the colon, but linalool was ineffective. Responses to AITC were reduced by the TRPA1 antagonist HC-030031 (100 μM), and were greatly diminished in Trpa1-/- duodenum and colon. Responses were not reduced by tetrodotoxin, 5-HT receptor antagonists, or atropine, but inhibition of prostaglandin synthesis reduced responses. Thus, functional TRPA1 channels are expressed by enterocytes of the duodenum and colon. Activation of enterocyte TRPA1 by food components has the potential to facilitate nutrient absorption.
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    Dietary advanced glycation end-products aggravate non-alcoholic fatty liver disease
    Leung, C ; Herath, CB ; Jia, Z ; Andrikopoulos, S ; Brown, BE ; Davies, MJ ; Rivera, LR ; Furness, JB ; Forbes, JM ; Angus, PW (BAISHIDENG PUBLISHING GROUP INC, 2016-09-21)
    AIM: To determine if manipulation of dietary advanced glycation end product (AGE), intake affects non-alcoholic fatty liver disease (NAFLD) progression and whether these effects are mediated via RAGE. METHODS: Male C57Bl6 mice were fed a high fat, high fructose, high cholesterol (HFHC) diet for 33 wk and compared with animals on normal chow. A third group were given a HFHC diet that was high in AGEs. Another group was given a HFHC diet that was marinated in vinegar to prevent the formation of AGEs. In a second experiment, RAGE KO animals were fed a HFHC diet or a high AGE HFHC diet and compared with wildtype controls. Hepatic biochemistry, histology, picrosirius red morphometry and hepatic mRNA were determined. RESULTS: Long-term consumption of the HFHC diet generated significant steatohepatitis and fibrosis after 33 wk. In this model, hepatic 4-hydroxynonenal content (a marker of chronic oxidative stress), hepatocyte ballooning, picrosirius red staining, α-smooth muscle actin and collagen type 1A gene expression were all significantly increased. Increasing the AGE content of the HFHC diet by baking further increased these markers of liver damage, but this was abrogated by pre-marination in acetic acid. In response to the HFHC diet, RAGE(-/-) animals developed NASH of similar severity to RAGE(+/+) animals but were protected from the additional harmful effects of the high AGE containing diet. Studies in isolated Kupffer cells showed that AGEs increase cell proliferation and oxidative stress, providing a likely mechanism through which these compounds contribute to liver injury. CONCLUSION: In the HFHC model of NAFLD, manipulation of dietary AGEs modulates liver injury, inflammation, and liver fibrosis via a RAGE dependent pathway. This suggests that pharmacological and dietary strategies targeting the AGE/RAGE pathway could slow the progression of NAFLD.
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    Selenium-Enriched Agaricus bisporus Mushroom Protects against Increase in Gut Permeability ex vivo and Up-Regulates Glutathione Peroxidase 1 and 2 in Hyperthermally-Induced Oxidative Stress in Rats
    Maseko, T ; Dunshea, FR ; Howell, K ; Cho, H-J ; Rivera, LR ; Furness, JB ; Ng, K (MDPI AG, 2014-06)
    Dietary effects of organic Se supplementation in the form of Se-enriched Agaricus bisporus mushroom on ileal mucosal permeability and antioxidant selenoenzymes status in heat induced oxidative stress in rats were evaluated. Acute heat stress (40 °C, 21% relative humidity, 90 min exposure) increased ileum baseline short circuit current (Isc; 2.40-fold) and epithelial conductance (Ge; 2.74-fold). Dietary supplementation with Se-enriched A. bisporus (1 µg Se/g feed) reduced (p < 0.05) ileum Isc and Ge during heat stress to 1.74 and 1.91 fold, respectively, indicating protection from heat stress-induced mucosal permeability increase. The expression of ileum glutathione peroxidase (GPx-) 1 and 2 mRNAs were up-regulated (p < 0.05) by 1.90 and 1.87-fold, respectively, for non-heat stress rats on the Se-enriched diet relative to the control. The interplay between heat stress and dietary Se is complex. For rats on the control diet, heat stress alone increased ileum expression of GPx-1 (2.33-fold) and GPx-2 (2.23-fold) relative to thermoneutral conditions. For rats on the Se-enriched diet, heat stress increased (p < 0.05) GPx-1 expression only. Rats on Se-enriched + α-tocopherol diet exhibited increased expression of both genes (p < 0.05). Thus, dietary Se-enriched A. bisporus protected against increase in ileum permeability and up-regulated GPx-1 and GPx-2 expression, selenoenzymes relevant to mitigating oxidative stress.
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    The gut as a sensory organ
    Furness, JB ; Rivera, LR ; Cho, H-J ; Bravo, DM ; Callaghan, B (NATURE PUBLISHING GROUP, 2013-12)
    The gastrointestinal tract presents the largest and most vulnerable surface to the outside world. Simultaneously, it must be accessible and permeable to nutrients and must defend against pathogens and potentially injurious chemicals. Integrated responses to these challenges require the gut to sense its environment, which it does through a range of detection systems for specific chemical entities, pathogenic organisms and their products (including toxins), as well as physicochemical properties of its contents. Sensory information is then communicated to four major effector systems: the enteroendocrine hormonal signalling system; the innervation of the gut, both intrinsic and extrinsic; the gut immune system; and the local tissue defence system. Extensive endocrine-neuro-immune-organ-defence interactions are demonstrable, but under-investigated. A major challenge is to develop a comprehensive understanding of the integrated responses of the gut to the sensory information it receives. A major therapeutic opportunity exists to develop agents that target the receptors facing the gut lumen.
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    Knock out of neuronal nitric oxide synthase exacerbates intestinal ischemia/reperfusion injury in mice
    Rivera, LR ; Pontell, L ; Cho, H-J ; Castelucci, P ; Thacker, M ; Poole, DP ; Frugier, T ; Furness, JB (SPRINGER, 2012-08)
    Recent investigation of the intestine following ischemia and reperfusion (I/R) has revealed that nitric oxide synthase (NOS) neurons are more strongly affected than other neuron types. This implies that NO originating from NOS neurons contributes to neuronal damage. However, there is also evidence of the neuroprotective effects of NO. In this study, we compared the effects of I/R on the intestines of neuronal NOS knockout (nNOS(-/-)) mice and wild-type mice. I/R caused histological damage to the mucosa and muscle and infiltration of neutrophils into the external muscle layers. Damage to the mucosa and muscle was more severe and greater infiltration by neutrophils occurred in the first 24 h in nNOS(-/-) mice. Immunohistochemistry for the contractile protein, α-smooth muscle actin, was used to evaluate muscle damage. Smooth muscle actin occurred in the majority of smooth muscle cells in the external musculature of normal mice but was absent from most cells and was reduced in the cytoplasm of other cells following I/R. The loss was greater in nNOS(-/-) mice. Basal contractile activity of the longitudinal muscle and contractile responses to nerve stimulation or a muscarinic agonist were reduced in regions subjected to I/R and the effects were greater in nNOS(-/-) mice. Reductions in responsiveness also occurred in regions of operated mice not subjected to I/R. This is attributed to post-operative ileus that is not significantly affected by knockout of nNOS. The results indicate that deleterious effects are greater in regions subjected to I/R in mice lacking nNOS compared with normal mice, implying that NO produced by nNOS has protective effects that outweigh any damaging effect of this free radical produced by enteric neurons.
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    Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor
    Callaghan, B ; Kosari, S ; Pustovit, RV ; Sartor, DM ; Ferens, D ; Ban, K ; Baell, J ; Nguyen, TV ; Rivera, LR ; Brock, JA ; Furness, JB (WILEY, 2014-03)
    BACKGROUND AND PURPOSE: Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. EXPERIMENTAL APPROACH: The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). KEY RESULTS: Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. CONCLUSIONS AND IMPLICATIONS: Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin.