Development of RTgutGC as a Tool for Fish Feed Development
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A rainbow trout intestinal epithelial cell line, RTgutGC, has been used in this thesis to investigate the effects of various stresses in aquaculture on the gastrointestinal (GI) tract and for identifying possible beneficial actions to take to protect gut health. RTgutGC cells were studied in four kinds of media. L15 with a supplement of fetal bovine serum (FBS) was the normal growth medium (L15/FBS). Serum starvation was done in the basal medium alone, L15. Deprivation of serum, amino acids, and vitamins was accomplished in L15/ex, which had only L-15 salts with galactose and pyruvate. Complete starvation of all nutrients was achieved in L15/salts. In these studies, cell viability was assessed with alamar Blue (AB) for metabolic activity and with carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for plasma membrane integrity. Confocal microscopy together with immunocytochemical (ICC) staining was used to evaluate microtubule organization and tight junctions and with fluorescent phalloidin staining to detect F-actin. Western blotting was used to measure specific protein levels, such heat shock protein 70 (HSP70). Intestinal epithelial barrier function was evaluated in a culture system in which RTgutGC monolayers divided a culture chamber into top and bottom wells and trans epithelial electrical resistance (TEER) and Lucifer Yellow (LY) permeability across the cell monolayers were measured. Intestinal epithelial wound healing and/or restitution was examined in a plastic fence assay in which cells formed monolayers on both sides of culture inserts that were then removed to form a gap or wound. Five general lines of investigation were explored on the topics of starvation, phytochemicals, antinutrionals, temperature, and phytochemical/temperature interactions. The results for each are summarized in the following five paragraphs. Rainbow trout intestinal epithelial cell monolayers survived serum starvation (L15), the deprivation of serum, amino acids, and vitamins (L15/ex), and the complete absence of nutrients (L15/salts) but some cellular activities and structures were altered, depending on the severity of the deprivation. During these three kinds of nutritional deprivation, most cells survived at least seven days as judged by their continued adherence to the plastic growth surface as observed by phase contrast microscopy and by cell viability measurements with AB and CFDA-AM. However, energy metabolism as measured with AB was diminished, especially in L15/salts with approximately an 85% decline. Under all types of nutrient deprivation, the cytoskeleton of cells remained intact. However, during nutritional deprivation, the actin stress fibers became thicker and many cells acquired circumferential fibers, especially in L15/salts. In L15/ex and L15/salts, the fibers of the alpha-tubulin network appeared thicker and longer and as well the microtubular organizing centres were larger and more intensely stained. ZO-1 (tight junction protein-1) was detected at the periphery of cells in monolayers, with nutritional deprivation causing only a slight change. However, in L15/salts epithelial barrier functions were impaired. In L15/FBS the TEER was around 30-40 Ω cm2 and approximately 75 % of the LY was retained in the top chamber. In L15/salts the TEER was approximately 15-20 Ω cm2 and only about 30 % of the LY was retained in the top chamber. Seven days after the creation of gaps in monolayers, the gaps were filled by RTgutGC cells in L15/FBS. As cells proliferate in L15/FBS as well as migrate, this is considered wound healing. By contrast, RTgutCG in L15 filled the gap much more slowly, and as most fish cells do not proliferate in L15 alone, the gap closing is considered to be due to cell migration alone or restitution. RTgutGC in L15/salts failed to migrate into gaps, but if after seven days L15/FBS was added, the cells did, emphasizing that they were still alive. When after seven days in either L15/FBS or L15/salts monolayers were trypsinized, the diameter and volumes of cells were respectively 17.9 ± 1.6 μm and 3.1 ± 0.8 pL for L15/FBS and 15.0 ± 0.3 μm and 1.8 ± 0.1 pL for L15/salts. Despite this reduction in size, when placed in L15/FBS, these cells could reattach to plastic and grow to form monolayers, emphasizing again that they were viable after seven days in L15/salts. Thus rainbow intestinal epithelial cells survived for at least seven days in the complete absence of nutrients but starvation impaired their barrier functions and ability to repair wounds. RTgutGC, a rainbow trout intestinal epithelial cell line, was used as an intestinal model to study the effects of naringenin (N), a plant secondary metabolite found in grapefruits with antioxidant, anti-inflammatory, and anti-carcinogenic properties. 30 and 100 μM N generated a flattened cell morphology with more defined cell borders. Most significant reductions in cellular viability were seen when incubated with 100 μM N, where in L15 medium, metabolic activity decreased by 59% and plasma membrane integrity decreased by 31%. However, lower concentrations of N had no effect on cellular viability. With the cytoskeleton still intact, N increased circumferential actin while decreasing the amount of stress fibers in the cells. Increasing concentrations of N caused a dose response increase in TEER. A significant reduction (39%) in monolayer permeability as measured by LY rejection assay was observed with 100 μM N. No changes in ZO-1 or claudin 3 staining was observed. Significant reductions in migration and restitution were observed with 50 and 75 μM N, but not lower concentrations. N did not cause any changes in HSP70 protein expression. The work, though in vitro, demonstrates the potential beneficial effects of N (concentration of 30 μM) as a possible feed additive. RTgutGC was used to study the effects of antinutritional factors (ANFs) on intestinal epithelial cell restitution. 100 μg/mL of Bowman-Birk inhibitor significantly (p < 0.05) reduced restitution where cells reached a total percent migration of 13 ± 3% (control = 23 ± 7%.). 0.75 and 2.25 μg/mL of wheat germ agglutinin (WGA) significantly (p < 0.01) reduced restitution at a total percent migration of 13% ± 3% and -11 ± 3% respectively (control = 49 ± 16%). Additionally, WGA caused the loss of actin stress fibers with actin being more peripherally located. 8 mM of butyrate significantly (p < 0.05) reduced restitution with cells reaching a total percent migration of 18 ± 6% (control = 39 ± 11%). Increasing vacuole formation was also observed with increasing concentrations of butyrate. Kunitz inhibitor and soybean agglutinin had little to no effect on RTgutGC restitution. For the first time, we have shown negative effects of ANFs on fish intestinal cell restitution and demonstrated preliminary uses of RTgutGC as an in vitro method to screen ANFs. The capacity of rainbow trout epithelial cells in L15/FBS to heal a wound whether through a combination of cell migration and proliferation or just cell migration (restitution) was profoundly influenced by temperature. Relative to the normothermic temperature of 18 °C for RTgutGC, a decrease in temperature to 4 oC caused a significant reduction in wound healing. Increasing the temperature to 26 oC slightly but not significantly increased wound healing. However, an increase to 32 oC (heat stress) during the wound healing assay caused the cell monolayer to shrivel up and die. Additionally, if cells were exposed to a heat stress temperature (32 oC) for 3 h before the start of wound healing, small numbers of individual migrating cells could be observed, not reaching full gap closure. The induction of a thermotolerant state by heat pre-conditioning (26 oC for 24 h) before heat stress (32 oC for 3 h) restored wound healing to a capacity similar to the non-heat stress control, with full gap closure seen within 3 days. The study suggests that cellular wound healing can be highly dependent on temperature and that preventative measures, such as heat pre-conditioning, can help mitigate the negative effects of heat stress on wound healing. Possible protective actions of N on the recovery of rainbow trout intestinal epithelial cells from heat stress were investigated but N only modestly improved recovery of cellular morphology. RTgutGC monolayers in either L15/FBS, L15 or L15/salts with or without N were subjected to heat stress of 32 °C for 1.5 or 3 h and returned to 18 °C to recover for 24 h. At one hour after the end of the heat treatments the monolayers were no longer confluent but had holes as a result of cells shrivelling and rounding. When the heat stress had been 1.5 h, the cellular morphology was largely restored 24 h later whether N was present or not. When the heat stress had been 3 h, N improved the adherence and shape of cells 24 h later, especially for recovery in L15/salts. However, with increasing N concentrations of up to 100 μM, no trend of improvement was observed in cell viability as measured with AB or CFDA-AM and 100 μM N did not alter the heat activation of caspase-3. Yet increasing N did alter the organization of F-actin during recovery. Stress fibers were diminished but circumferential actin became more pronounced with increasing N. This was true whether the cells were in L15/FBS or L15/salts but more evident in L15/salts. Thus the reorganization F-actin to the cell periphery might have improved the recovery of cell shape after a heat stress. Naringenin failed to modulate HSP70 levels in cultures at 18 °C whether the cultures were recovering from a heat stress or not. Therefore, HSP70 was unlikely to be mediating the improvement by N in the recovery of cell shape from heat stress.
Cite this work
Patrick Gilles Pumputis (2017). Development of RTgutGC as a Tool for Fish Feed Development. UWSpace. http://hdl.handle.net/10012/12150