The Impact of Acute Matriptase Inhibition in Hepatic Inflammatory Models

Purpose. Dysfunction of matriptase-2 can be involved in iron regulatory disorder via downregulation of hepcidin expression. In the present study, we investigated the effects of 3-amidinophenylalanine-derived matriptase inhibitors on porcine hepatic inflammatory cell models. Methods. Hepatocyte-Kupffer cell cocultures (ratio of 2 : 1 and 6 : 1) were treated with four structurally related matriptase inhibitors at 50 μM. Cell cytotoxicity and relative expressions of IL-6 and IL-8 and the levels of hepcidin were determined by MTS and porcine-specific ELISA. The extracellular H2O2 contents were analyzed by Amplex Red method. Results. Matriptase inhibitors at 50 µM for 24 h did not increase cell death rate. The elevated ROS production observed after short-term application of inhibitor MI-441 could be correlated with lowered hepcidin expression. MI-460 could significantly enhance hepcidin levels in the supernatants of cocultures (by 62.21±26.8% in hepatocyte-Kupffer cell, 2 : 1, and by 42.6±14.3% in hepatocyte-Kupffer cell, 6 : 1, cocultures, resp.). No significant changes were found in IL-6 and IL-8 levels in cocultures exposed to matriptase inhibitors. Conclusions. Based on in vitro findings, administration of MI-460 via modulation of hepcidin expression without cytotoxic and oxidative stress inducing properties might be a reliable alternative to treat iron overload in human and veterinary clinical practice

The Impact of Acute Matriptase Inhibition in Hepatic Inflammatory Models

Purpose. Dysfunction of matriptase-2 can be involved in iron regulatory disorder via downregulation of hepcidin expression. In the present study, we investigated the effects of 3-amidinophenylalanine-derived matriptase inhibitors on porcine hepatic inflammatory cell models. Methods. Hepatocyte-Kupffer cell cocultures (ratio of 2 : 1 and 6 : 1) were treated with four structurally related matriptase inhibitors at 50 μM. Cell cytotoxicity and relative expressions of IL-6 and IL-8 and the levels of hepcidin were determined by MTS and porcine-specific ELISA. The extracellular H2O2 contents were analyzed by Amplex Red method. Results. Matriptase inhibitors at 50 µM for 24 h did not increase cell death rate. The elevated ROS production observed after short-term application of inhibitor MI-441 could be correlated with lowered hepcidin expression. MI-460 could significantly enhance hepcidin levels in the supernatants of cocultures (by 62.21±26.8% in hepatocyte-Kupffer cell, 2 : 1, and by 42.6±14.3% in hepatocyte-Kupffer cell, 6 : 1, cocultures, resp.). No significant changes were found in IL-6 and IL-8 levels in cocultures exposed to matriptase inhibitors. Conclusions. Based on in vitro findings, administration of MI-460 via modulation of hepcidin expression without cytotoxic and oxidative stress inducing properties might be a reliable alternative to treat iron overload in human and veterinary clinical practice

Effect of dietary cereal type, crude protein and butyrate supplementation on metabolic parameters of broilers

This study investigates the metabolic effects of maize- or wheat-based diets with normal (NP) and lowered (LP) dietary crude protein level [the latter supplemented with limiting amino acids and sodium (n-)butyrate at 1.5 g/kg diet] at different phases of broiler fattening. Blood samples of Ross 308 broilers were tested at the age of 1, 3 and 6 weeks. Total protein (TP) concentration increased in wheat-based and decreased in LP groups in week 3, while butyrate reduced albumin/TP ratio in week 1. Uric acid level was elevated by wheat-based diet in week 1 and by wheat-based diet and butyrate in week 3, but decreased in LP groups in weeks 3 and 6. Aspartate aminotransferase activity was increased by wheat-based diet in week 3, and creatine kinase activity was intensified by LP in weeks 3 and 6. Blood glucose level decreased in wheat-based groups in week 3; however, triglyceride concentration was augmented in the same groups in week 3. No change of glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide and insulin concentration was observed. In conclusion, an age-dependent responsiveness of broilers to dietary factors was found, dietary cereal type was a potent modulator of metabolism, and a low crude protein diet supplemented with limiting amino acids might have a beneficial impact on the growth of chickens

Effects of butyrate on the insulin homeostasis of chickens kept on maize- or wheat-based diets

The aim of the present study was to investigate the effects of butyrate as a feed supplement on the expression of insulin signalling proteins as potent regulators of metabolism and growth in Ross 308 broiler chickens fed maize- or wheat-based diets. Both diets were supplemented with non-protected butyrate (1.5 and 3.0 g/kg of diet, respectively) or with protected butyrate (0.2 g/kg of diet); the diet of the control groups was prepared without any additives (control). On day 42 of life, systemic blood samples were drawn for analyses of glucose and insulin concentrations, and tissue samples (liver, gastrocnemius muscle and subcutaneous adipose tissue) were taken for Western blotting examinations. The expression of key insulin signalling proteins (IRβ, PKCζ and mTOR) was assessed by semiquantitative Western blotting from the tissues mentioned. The type of diet had a remarkable influence on the insulin homeostasis of chickens. The wheat-based diet significantly increased IRβ and mTOR expression in the liver as well as mTOR and PKCζ expression in the adipose tissue when compared to animals kept on a maize-based diet. IRβ expression in the liver was stimulated by the lower dose of non-protected butyrate as well, suggesting the potential of butyrate as a feed additive to affect insulin sensitivity. Based on the results obtained, the present study shows new aspects of nutritional factors by comparing the special effects of butyrate as a feed additive and those of the cereal type, presumably in association with dietary non-starch polysaccharide- (NSP-) driven enteric shortchain fatty acid release including butyrate, influencing insulin homeostasis in chickens. As the tissues of chickens have physiologically lower insulin sensitivity compared to mammals, diet-associated induction of the insulin signalling pathway can be of special importance in improving growth and metabolic health

A butirát epigenetikus és a mikroszomális méregtelenítő enzimekre gyakorolt hatásának vizsgálata a csirke májában

The short chain fatty acid butyrate is one of the major end products of the anaerobic microbial fermentation of carbohydrates in the forestomachs of ruminants and in the large intestine of monogastric mammals, birds and humans. Butyrate is also a widely used feed additive as an alternative growth promoter due to its beneficial effects on growth performance, first of all in poultry and pig nutrition. This is of special importance due to the banning of the traditional antibiotic growth promoters in the European Union. Butyrate may provoke its effects on metabolism via many different, yet not completely defined pathways. One of those pathways is that butyrate is known as a histone deacetylase inhibitor, inducing histone hyperacetylation in vitro and playing a predominant role in the epigenetic regulation of gene expression and cell function. It was hypothesized in this study that butyrate, applied as feed additive, might cause similar in vivo modifications in the chromatin structure of the hepatocytes of chickens in the early post-hatch period. Further, it could influence the expression of certain genes and therefore modify the activity of hepatic microsomal drugmetabolizing cytochrome P450 (CYP) enzymes, resulting in pharmacoepigenetic interactions with simultaneously applied xenobiotics. Most experiments of this PhD study were performed in broiler chickens, because chickens are not only target species of butyrate administration as feed additive, but they can also serve as model for the investigation of butyrate’s actions. Regarding the intestinal effects of butyrate, this study aimed to compare the influence of butyrate on small intestinal histomorphology in chicken and rat, the latter as a model animal for monogastric mammals. In vivo studies were carried out in chicken to investigate the molecular mechanisms of butyrate’s epigenetic actions on the liver. Broiler chicks in the early post-hatch period received butyrate-supplemented diet (1.5 g/kg diet) or were treated once daily with orally administered bolus of butyrate following overnight fasting with two different doses (0.25 or 1.25 g/kg body weight per day) for five days. After slaughtering, cell organelles were separated by differential centrifugation from the livers and acetylation of hepatic core histones was screened from cell nuclei by western blotting. Effects of butyrate on CYP gene expression were tested at first in vitro on primary culture of chicken hepatocytes, followed by an in vivo trial with butyrate-fed chickens. The activity of the most important CYP enzymes was also monitored by aminopyrine N-demethylation, aniline hydroxylation and testosterone 6β-hydroxylation assays from the microsomal fractions of chickens. Furthermore, the interaction of butyrate and the macrolide antibiotic erythromycin was tested in vitro and finally also in vivo by studying the major pharmacokinetic parameters of erythromycin

Molecular effects of intermittent stress on primary feline uroepithelial cell culture as an in vitro model of feline idiopathic cystitis

IntroductionThe most common cause of feline lower urinary tract disease (FLUDT) is feline idiopathic cystitis (FIC), which is a complex multifactorial disease with symptoms including stranguria, dysuria, hematuria, and pain during urination. The development of these symptoms is often triggered by stress, and in case of chronic stress, these symptoms will many times return. One of the most important stress hormones in the pathogenesis of FIC is norepinephrine (NE), as persistently elevated level of this hormone can be measured in the blood of cats with FIC. However, it is not well understood if recurrently elevated level of NE has any direct effect on urinary bladder, therefore the aim of this study was to investigate the molecular effects of intermittent NE exposure on feline primary uroepithelial cell culture.MethodsPrimary uroepithelial cells were gained from the mucosa of the bladder of a euthanized cat, and were cultured for 6 days, then they were exposed to 10, 100, and 1,000 μM NE treatment for 3 × 1 h, including a 1 h long regeneration period between exposures.ResultsNE was able to trigger pro-inflammatory response and oxidative stress in the uroepithelial cells by increasing the level of stromal cell derived factor 1 (SDF-1) and H2O2 in cell culture media. In addition, NE increased the permeability of the uroepithelium, since decreased glycosaminoglycan (GAG) concentration, tight junction protein claudin-4 content, and TER values were measured after the NE treatments.DiscussionBased on these results it can be concluded that recurrent stress mimicked by 3×1 h NE treatment has a direct molecular effect on the uroepithelial cells, which leads to inflammatory response, oxidative stress and decreased barrier function of the uroepithelium. Therefore, intermittent release of NE may have an important role in the pathogenesis of FIC and the results of this study may contribute to a better understanding of the development of this illness

Role of incretin hormones in the regulation of insulin homeostasis and carbohydrate metabolism Literature review

Summary In the present literature review, the authors discuss the central role of the gutdriven incretin hormones, namely GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide 1), as members of the enteroinsular axis, with regard on the pancreatic insulin production. The GIP, produced by K cells of the small intestines after feed intake, stimulates the insulin secretion of β cells mostly by the mediation of cAMP as a second messenger. Further, GLP-1, released from L cells, being presented in both small and large intestines, increases insulin synthesis by affecting insulin gene expression, enhances pancreatic insulin exocytosis and stimulates proliferation as well as differentiation of β cells. In addition, glucagon production of α cells can be inhibited by GLP-1, while GIP is capable to increase pancreatic glucagon secretion. Concerning the incretin effects in further tissues, lipogenesis and proliferation of osteoblasts can be also stimulated by GIP, leading to enhanced lipid storage in adipose tissue and to faster bone formation of growing animals, respectively. The way of incretin action is partly differing in birds from that of mammals, increasing pancreatic insulin release more likely by influencing the somatostatin production of δ cells rather than by direct stimulation of β cells. However, their anorexigenic effect, similarly to mammalian species, was stated in chicken, as well. Based on the available literature data, the authors declare that special emphasis should be taken on the role of incretin hormones in the complex regulation of insulin secretion. Influencing incretin and insulin homeostasis by nutrition or certain drugs can be of special relevance in the treatment of diabetic patients, and, based on insulin’s anabolic action, in improving the growth performance of food-producing animals

The effects of T-2 toxin on animal health, focusing especially on poultry - Literature review

SUMMARY In the present literature review, the authors discuss the various general and specific effects of the T-2 toxin on animal health, focusing on poultry. T-2 toxin is one of the most toxic trichothecene mycotoxin produced by various Fusariumspecies. These are destructive pathogens on cereal crops and other commodities and produce the toxins before and immediately after harvesting. Although most of the bird species are quite resistant to the various mycotoxins, the issue of T-2 toxin contamination has a significant and remarkable importance in the poultry farming and production worldwide. The main targets of the toxin are the rapidly proliferating and differentiating cells and tissues with a high protein turnover, including the small intestines, the liver and immune cells. The toxic effects exerted by T-2 toxin include the inhibition of DNA, RNA and protein synthesis and it can affect the production of immunoglobulins or the humoral immunity as well. These effects can simply lead to immunosuppression, consequently the animals will be highly susceptible to infections caused by facultative pathogens. T-2 toxin furthermore generates reactive oxygen species (ROS) that induce lipid peroxidation which in turn leads to changes in membrane integrity, cellular redox signaling and the antioxidant status of the cells. The detection of the problem can be really difficult, since in numerous cases we can talk about subacute exposure for a longer time. It can affect the productivity of the animals in a negative way, but it does not lead to specific and well recognizable symptoms. As genotoxic and cytotoxic effects indicate that T-2 toxin is highly toxic on animal health, and as it is widespread in cereals and food, profound research of its toxic potential in animals is necessary in the future